Serological Evidence of Ongoing Transmission of Dengue Virus in Permanent Residents of Key West,Florida

Abstract

Introduction:

Dengue fever is the most important mosquito-borne viral disease in the world, with 40% of the global population at risk of infection. Dengue virus is responsible for infections in over 100 countries, including the Americas and Caribbean Basin; however, it has been largely eradicated from the United States through the implementation of effective vector control programs. However, between 2009 and 2010, 27 permanent residents of Key West, Florida, were reported to have locally acquired infections, marking the first autochthonous cases detected in Florida since 1934. Despite this recent and unusual transmission, and the potential risk of serious illness associated with sequential infections, no active surveillance had been conducted since.

Materials and Methods:

A serosurvey of permanent residents of Key West, Florida, was conducted in March of 2012. After informed consent, enrolled participants (n=173) were given a dengue virus rapid diagnostic test and completed a corresponding questionnaire.

Results:

The presence of immunoglobulin G (IgG) antibodies was indicated in 12 participants (6.9%), all of whom reported travel to endemic countries within the past 2 years. Surprisingly, six participants (3.5%) without any recent travel outside the state of Florida gave positive results for IgM antibodies. The presence of birdbaths and bromeliads on the property and sleeping outdoors emerged as significant factors related to previous exposure, whereas home air conditioning without the use of open windows and the use of mosquito repellent were protective.

Conclusions:

These findings suggest local transmission occurred in Key West in early 2012, indicating that transmission may not have subsided in 2010.

Introduction

Dengue fever is the most important mosquito-borne viral disease in the world, with 40% of the global population at risk of infection and a projected 5.2 billion individuals to be at risk by 2085 (Knowlton et al. 2009; World Health Organization 2012). Currently, dengue virus (DENV) is endemic in over 100 countries spanning the Americas, Caribbean Basin, Asia, and Africa, with 50–100 million cases of dengue fever, 250,000 cases of severe sequential infections including dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), and 25,000 deaths reported each year (Wilder-Smith and Schwartz 2005, Tomashek 2011, World Health Organization 2012). Historically, the disease had a widespread global distribution; however, it was largely eradicated from the United States through the implementation of effective vector control programs (Gubler 1998, Bouri et al. 2012). From July to October of 2009, 27 residents of Key West, Florida, were reported to have locally acquired DENV infections, marking the first autochthonous cases detected in Florida since 1934 (Ehrenkranz et al. 1971, Centers for Disease Control and Prevention 1999, 2010).

After only three of these locally acquired cases were reported, the Florida Department of Health conducted a seroprevalence survey employing a clustered door-to-door sampling technique that began at the households of index cases, in Old and Mid-Town Key West, Florida (Radke et al. 2012). Because this serosurvey was conducted before the height of the 2009 outbreak and the 63 additional cases reported the following year, the total number of infected individuals was likely underestimated (Centers for Disease Control and Prevention 2010). Because no other serological studies had been conducted in Key West to date, a postepidemic estimate of the overall population at risk for sequential infections was not known. This information is critical considering the consequences of sequential DENV infections, which are associated with more severe symptoms such as DHF, DSS, and death (Guzman et al. 2010). Besides leading to poor health outcomes, these sequential infections can also lead to high medical costs and could have a significant negative impact on local tourism (Tapasvi et al. 2010, Shepard et al. 2011). To determine if transmission was ongoing, a convenience sample of permanent residents was screened for recent infections with a rapid test and given a corresponding demographic and exposure related questionnaire.

Materials and Methods

During March, 2012, enrollment of study participants was conducted in the parking lots of major regional grocery store chains (Publix Super Markets, Albertsons, Winn-Dixie) in Key West, Florida. All individuals that approached grocery stores during enrollment periods were invited to participate in the study. Enrollment criteria constrained the study population to those over the age of 18 years who lived or worked on the island for at least 7 months per year. Every study participant provided written informed consent under a protocol approved by the Western Institutional Review Board prior to participation.

The presence of immunoglobulin M (IgM) or IgG antibodies from a participant blood sample was assessed using a qualitative immunochromatographic assay (Dengue Virus IgG/IgM Whole Blood/Serum/Plasma RapiCard^™ InstaTest from Cortez Diagnostics, Inc., Calabasas, CA). This rapid test method uses a plastic test cartridge containing a nitrocellulose membrane coated with two bands of mouse anti-human IgM- and IgG-binding proteins. After addition of the plasma, blood, or serum sample, human IgM and IgG antibodies complex with a colored recombinant DENV-specific antigen–colloidal gold conjugates and migrate to the test window, forming a maroon line after binding to their respective binding proteins. This rapid test methodology has been demonstrated to be 99–100% sensitive and 96% specific for detecting DENV infections and is able to simultaneously detect both recent (IgM) and previous (IgG) DENV antibodies (Sang et al. 1998, Vaughn et al. 1998, Porter et al. 1999). Tests were conducted according to manufacturer's instructions. Briefly, a finger prick blood sample was taken from each participant, applied to the test well along with two drops of the provided buffer, and allowed to incubate at ambient temperature (80–85°F) for 20 min. Positive results appear as maroon lines along the IgM and/or IgG bands, and negative results appear as a maroon line present at the control line only. All participants with a positive response were confirmed with a second test.

In addition to the rapid test, participants completed an enrollment questionnaire in which they reported their home and work addresses, demographic information, travel history, as well as various indicators of mosquito exposure. With the data acquired from these questionnaires, univariate and multivariate logistic regression were conducted in Stata 12.0 (StataCorp, College Station, TX) to investigate which factors may have influenced the likelihood of previous infection for residents participating in the study. Spatial distribution of respondent addresses was assessed with ArcGIS 10.0 (Esri, Redlands, CA).

Results

The current study enrolled a subset of 173 Key West residents that were reasonably representative of the general population with respect to age, gender, and ethnicity when compared to the 2010 US Census and the previous (2009 serosurvey by Radke et al.) (Table 1) (US Department of Commerce 2010, Radke et al. 2012). Respondents' home and work addresses were evenly dispersed across the entire island, further suggesting a successful recruitment of individuals representing the overall resident population in Key West (Fig. 1). Screening with the rapid diagnostic test identified 12 IgG-positive participants (6.9%). Unexpectedly, the presence of IgM antibodies was indicated in six participants (3.5%). None of the participants were positive with both a recent (IgM) and previous (IgG) infection.

FIG. 1.

Study enrollment locations (green circles), immunoglobulin-negative respondents (yellow circles), as well as the immunoglobulin-positive respondents for both immunoglobulin M (IgM) (red) and IgG (blue) appear with their reported home (squares) and work (triangles) addresses with respect to the roadways and public parcels. Color images available online at www.liebertpub.com/vbz

Table 1.

Demographic Information of Key West, Florida, Residents Comparing Participants in the 2009 and 2012 Serosurveys and the Associated 2010 Census Report

	Current study	2009 study (Radke et al. 2009)	(U.S. Department of Commerce, 2010) Census reports
Median age (age range)	52 (18–84)	53 (15–95)	41.5 (all ages)
Age groups
18–35	21.21%	—	27.6%^a
35–50	22.42%	—	24.00%
50–60	29.70%	—	16.00%
60+	26.67%	—	20.20%
Gender
Male	52.38%	58%	55.40%
Female	47.62%	42%	44.60%
Race
White	79.04%	78%	66.10%
Black	7.78%	—	9.00%
Hispanic	9.58%	—	21.20%
Other/multiple	3.59%	—	3.70%

Age group spans 15–35.

Univariate analysis of the survey data uncovered several significant factors associated with each type of seropositivity (Table 2). For IgM-positive individuals, central air conditioning use in the home was protective against infection (odds ratio [OR]=0.11). The greatest correlates for recent infection were sleeping outdoors once per week (OR=14.70) and the self-reported presence of birdbaths (OR=10.17) or bromeliads (OR=10.98) on residential property. All IgM-positive individuals recalled not using repellent on themselves or being bitten at home and were between the ages of 18 and 80. None of the IgM-positive individuals reported leaving the state of Florida in the previous 12 months, suggesting that their exposure to DENV occurred locally and not in an endemic region.

Table 2.

Logistic Regression Analysis Results from Serosurvey Questionnaire Data

	Univariate			Multivariate
Variable	Odds ratio	p value	95% Confidence interval	Odds ratio	p value	95% Confidence interval
IgM
Home air conditioning	0.11	0.038^a	(0.01, 0.89)	0.09	0.038^a	(0.01, 0.87)
Sleep outdoors once per week	14.70	0.040^a	(1.14, 190.27)	55.72	0.020^a	(1.90, 1637.09)
Birdbath on property	10.17	0.008^a	(1.85, 56.02)	9.31	0.020^a	(1.43, 60.74)
Bromeliads on property	10.98	0.031^a	(1.24, 96.95)	9.97	0.041^a	(1.10, 90.68)
Miss work >10 days with fever	6.50	0.045^a	(1.05, 40.43)	5.96	0.102	(0.70, 50.74)
IgG
Work air conditioning	0.08	0.010^a	(0.01, 0.54)	0.09	0.016^a	(0.01, 0.64)
Window cooling at work	5.44	0.036^a	(1.11, 26.54)	4.97	0.057	(0.95, 25.90)
Work outside (absence of screens)	6.50	0.057	(0.94, 44.80)	6.41	0.062	(0.91, 45.26)
Rash in last 12 months	6.20	0.004^a	(1.82, 21.13)	5.82	0.006^a	(1.67, 20.30)
Any (IgM+IgG)
Work air conditioning	0.16	0.032^a	(0.31, 0.86)	0.17	0.045^a	(0.03, 0.96)
Window cooling at work	4.26	0.016^a	(1.31, 13.89)	3.79	0.036^a	(1.09, 13.18)
Sleep outdoors once per week	18.27	0.021^a	(1.56, 213.58)	44.75	0.009^a	(2.55, 784.22)
Using repellent on self	0.23	0.058	(0.05, 1.05)	0.24	0.066	(0.05, 1.10)
Rash in last 12 months	2.95	0.051	(0.99, 8.76)	2.84	0.067	(0.93, 8.65)

Variables found to be significant at alpha=0.05.

IgM, immunobgloublin M; IgG, immunoglobulin G.

In contrast to IgM-positive respondents, all IgG positives reported travel to a dengue-endemic area in the previous 2 years, suggesting that individuals could have acquired infection during travel. IgG positives had a narrower age range (37–83) than IgM-positive respondents. Univariate analyses suggested that having a work environment cooled by central air conditioning was protective against infection (OR=0.08). Factors that increased the likelihood of having an IgG-positive result included using open windows to cool the work environment (OR=5.44) and experiencing a rash within the previous 12 months (OR=6.20).

Additionally, IgM- and IgG-positive individuals were pooled into a single group, and univariate analysis was repeated. Similar to analyses of only IgG-positive individuals, the likelihood of having a previous infection was reduced when using central air conditioning in the work environment (OR=0.16). In contrast, the likelihood of previous infection increased when work areas were cooled by open windows (OR=4.26) or respondents reported sleeping outdoors once per week (OR=18.27). All seropositive individuals recalled being bitten by a mosquito in the last 12 months. The use of repellent before going outdoors was confirmed to be protective (OR=0.23), whereas those abstaining from the use of repellent had an increased likelihood of being previously infected (OR=4.29). In this study, only two of 18 positive participants (11%) reported applying repellent before going outdoors. After adjustment for both gender (not significant) and age (significant), similar ORs and levels of significance were found for factors associated with previous infection (Table 2).

The relatively small number of IgM- or IgG-positive participants precluded a more formal cluster analysis; however, the illustration of the locations of the home and work addresses provided some insight that was not previously available. Because the previous serosurvey was conducted only 200–1000 meters proximal to the reported cases in Old Town, located in the West side of the island, there was no direct evidence of transmission in permanent residents beyond Old Town. Using a more geographically diverse sample, the distribution of rapid test positive participants indicated that IgM-positive participants largely resided in the New Town (eastern) portion of Key West (Fig. 1), suggesting a wider spatial distribution that was previously documented (Centers for Disease Control and Prevention 2010, Radke et al. 2012). In contrast to IgM positives, IgG-positive participants more frequently resided in the Old and Mid-Town areas of Key West, which is consistent with the distribution of cases in the 2009 and 2010 outbreak (M.S. Doyle and A.L. Leal, pers. commun.). The work addresses of seropositive respondents were distributed in both the New and Old Town portions of the island and showed no obvious spatial pattern.

Discussion

The previous serosurvey in 2009 identified 3% (6/240) of Old Town residents to be IgM positive, indicating recent DENV infections (within 3 months), and 6% (16/240) IgG positive, indicating an infection prior to this 3-month interval (Radke et al. 2012). Despite the lack of reported cases in 2011, this study identified 3.5% IgM-positive (6/173) and 6.9% IgG-positive (12/173) full-time residents, indicating the possibility that Key West experienced an undetected outbreak in early 2012 of similar or greater magnitude than in 2009. The majority of results of the current (2012) serosurvey are in accord with the previous (2009) serosurvey, with air conditioning being protective and open window cooling, recalling mosquito bites, and birdbaths on the property increasing the likelihood of infection (Radke et al. 2012). In addition, the current study also found sleeping outdoors once per week, evidence of a rash within the preceding 12 months, and working outside increased the likelihood of previous infection. With none of the IgM-positive participants reporting recent travel outside Florida, the recollection of being bitten at home and the increased likelihood of infection associated with the presence of mosquito breeding sites on residential property (bromeliads, birdbaths, etc.), it appears that exposure to DENV in the early months of 2012 likely occurred in Key West.

The spatial pattern of IgG-positive home addresses could be indicative of exposure to DENV in the preceding 2009 and 2010 outbreaks because infections were limited mostly to the Old and Mid-Town regions of the island. Because all IgG-positive individuals reported travel to dengue-endemic countries, it is impossible to claim that the seropositive results of these individuals were a direct result of infection during the previous 2009 and 2010 outbreaks in Key West. It is not unreasonable to assume that DENV transmission terminated in 2009 and later re-started in 2010 due to reintroduction of the virus. Assuming DENV was not endemic to Florida prior to this outbreak, two scenarios for DENV introduction are possible. First, the virus could have been introduced by infected mosquitoes, and/or second, it could have been introduced by infected humans acting as carriers, thus delivering the virus upon arrival to the city. DENV introductions by infected mosquitoes traveling to susceptible areas via aircraft is unlikely when compared to the introductions by viremic human travelers, which are considered the most likely source of DENV introductions around the world (Tatem et al. 2006, Halstead 2008). Given the high incidence of DENV in the Caribbean and Latin America during the epidemic years and because Key West receives over two million visitors each year, it is possible that an infected traveler or returning resident brought the virus to Key West (Leeworthy et al. 2010, Key West Chamber of Commerce 2013).

Whereas the presence of IgM-positive participants with no travel history outside of Florida supports the notion of local transmission of DENV in early 2012, because other serological methods such as enzyme-linked immunosorbent assay (ELISA) were not employed, these results can only provide evidence of DENV circulation in 2012. All respondents who were seropositive using the rapid test were retested to increase the validity of the results and positive test results did correspond with previously documented risk factors; however, further studies should be conducted to determine the risk posed to the residents and visitors to Key West. Aside from these limitations, we believe this study was able to gain valuable insight into the nonseasonal residential population's likelihood of previous DENV infection. The use of exclusion criteria formulated to ensure that only year-round residents were enrolled barred the sizable seasonal elderly population that travels and resides in southern Florida during winter months (Smith and House 2006). By sampling at a variety of grocery store chains with large spectrums of clients, a wide range of socioeconomic classes, age groups, and broader spatial distribution of individuals was achieved compared to traditional door-to-door surveys (Carpenter and Moore 2006). Similar to the previous 2009 serosurvey, which found a median age of 53 (range 15–95), the median age of participants enrolled in this study was 52 years (range 18–84) (Radke et al. 2012). Despite the smaller sample size of the current study, it was as representative of the ethnicity, gender, and age of the resident population as the previous (2009) serosurvey (US Department of Commerce 2010, Radke et al. 2012).

Conclusions

The identification of IgM-positive permanent residents with no recent travel history outside of Florida supports the likelihood of autochthonous transmission in Key West in the early months of 2012. Further research with more specific serological methods such as ELISA are required to confirm if DENV was still circulating in 2012 and to determine if the same serotype and strain that caused the 2009 and 2010 outbreaks is still present. Given the volume of travelers from dengue-endemic countries that could introduce multiple serotypes of DENV to the island and the risk of sequential infections in permanent residents, these findings should encourage future surveillance and continuation of effective vector control strategies.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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