Dengue is a potentially fatal acute febrile illness (AFI) caused by four mosquito-transmitted dengue viruses (DENV-1–4) that are endemic in Puerto Rico. In January 2010, the number of suspected dengue cases reported to the passive dengue surveillance system exceeded the epidemic threshold and an epidemic was declared soon after.
To characterize the epidemic, surveillance and laboratory diagnostic data were compiled. A suspected case was a dengue-like AFI in a person reported by a health care provider with or without a specimen submitted for diagnostic testing. Laboratory-positive cases had: (i) DENV nucleic acid detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in an acute serum specimen; (ii) anti-DENV IgM antibody detected by ELISA in any serum specimen; or (iii) DENV antigen or nucleic acid detected in an autopsy-tissue specimen. In 2010, a total of 26,766 suspected dengue cases (7.2 per 1,000 residents) were identified, of which 46.6% were laboratory-positive. Of 7,426 RT-PCR-positive specimens, DENV-1 (69.0%) and DENV-4 (23.6%) were detected more frequently than DENV-2 (7.3%) and DENV-3 (<0.1%). Nearly half (47.1%) of all laboratory-positive cases were adults, 49.7% had dengue with warning signs, 11.1% had severe dengue, and 40 died. Approximately 21% of cases were primary DENV infections, and 1–4 year olds were the only age group for which primary infection was more common than secondary. Individuals infected with DENV-1 were 4.2 (95% confidence interval [CI]: 1.7–9.8) and 4.0 (95% CI: 2.4–6.5) times more likely to have primary infection than those infected with DENV-2 or -4, respectively.
This epidemic was long in duration and yielded the highest incidence of reported dengue cases and deaths since surveillance began in Puerto Rico in the late 1960's. This epidemic re-emphasizes the need for more effective primary prevention interventions to reduce the morbidity and mortality of dengue.
Dengue is a potentially fatal acute febrile illness that is endemic throughout the tropics and sub-tropics. Dengue has been endemic in Puerto Rico for several decades and recent epidemics occurred in 1994–5, 1998 and 2007. In January 2010, dengue surveillance indicated that an epidemic had begun. The epidemic peaked in early August and ended in December with a total of 26,766 suspected dengue cases identified, of which 128 were fatal. The 2010 epidemic was one of the longest in Puerto Rico history and resulted in the greatest number of cases and deaths ever detected. We analyzed the epidemiologic and immunologic characteristics of laboratory-confirmed dengue cases and age group-specific attack rates, and determined the frequency of first DENV infection and DENV-types among persons experiencing their first infection. This analysis indicated that 10–19 year-olds were most affected during the epidemic, and that DENV-1 was roughly four times more likely to be associated with clinically apparent illness upon first DENV infection than were DENV-2 or -4. The 2010 dengue epidemic demonstrated the heavy burden of illness due to dengue in Puerto Rico, re-emphasizing the critical need for effective primary prevention tools to reduce the morbidity and mortality due to dengue worldwide.
Citation: Sharp TM, Hunsperger E, Santiago GA, Muñoz-Jordan JL, Santiago LM, et al. (2013) Virus-Specific Differences in Rates of Disease during the 2010 Dengue Epidemic in Puerto Rico. PLoS Negl Trop Dis 7(4): e2159. doi:10.1371/journal.pntd.0002159
Editor: Justin V. Remais, Emory University, United States of America
Received: September 8, 2012; Accepted: February 26, 2013; Published: April 4, 2013
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: This investigation was funded by the US Centers for Disease Control and Prevention and Puerto Rico Department of Health. The funders contributed to all study stages, including investigation design, data collection and analysis, decision to publish, and preparation of the report.
Competing interests: The authors have declared that no competing interests exist.
Dengue virus (DENV) transmission is endemic throughout most of the tropics and sub-tropics and is estimated to result in ~50 million symptomatic infections and ~20,000 deaths each year , . Infection with any DENV can result in dengue, an illness characterized by fever, headache, retro-orbital eye pain, myalgia and rash . In some cases, dengue can progress to severe dengue , which includes dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS)  and is characterized by thrombocytopenia, increased vascular permeability with plasma leakage, severe organ involvement, and/or clinically significant bleeding . Supportive care with appropriate intravascular volume repletion has been shown to lower mortality associated with severe dengue .
The four related but serotypically distinct DENV-types, DENV-1, -2, -3 and -4, are transmitted by Aedes aegypti or Ae. albopictus mosquitoes , . Following infection, individuals develop short-lived, heterotypic immunity and long-lived, type-specific immunity . Primary infection is an individual's first DENV infection, and secondary infection is any subsequent infection with a DENV-type different from the first. Severe dengue is more common upon secondary infection ,  and may be affected by the order in which an individual is infected with the respective DENV-types , . Thus, increases in the force of DENV infection can result in a decrease in the age of primary and secondary infection . Both local patterns of circulation of the four DENV-types and force of infection can influence the age groups most affected by dengue and severe dengue.
The unincorporated United States territory of Puerto Rico is composed of 78 municipalities, an area of 3,515 square miles, and a population of 3,725,789 . The demographics of Puerto Rico are similar to the United States as median age is 36 years and 78.6% are white, although 99% are self-described Hispanic . Since the mid-1990's the health care system in Puerto Rico has included both public and private health care services, and dengue has been a reportable condition for several decades. Ae. aegypti is the predominant DENV vector on the island.
Dengue was first described in Puerto Rico in 1915  and outbreaks have been recognized since 1963 , . DHF was first reported in 1975 , , all four DENV-types have been identified on the island since 1982 , , and the first confirmed dengue-related death was reported in 1986 . Recent epidemics were detected in 1994–1995, 1998 and 2007, with 24,700 , 17,000  and 10,508  reported suspect cases, respectively (Table S1). During both epidemic and non-epidemic periods, 10–19 year olds have been the most affected age group for several decades.
In the present investigation, we describe a dengue epidemic that occurred in 2010, including differences in the epidemiology of cases infected with different DENV-types with respect to primary versus secondary infection.
Materials and Methods
A retrospective analysis of suspected dengue cases reported to surveillance systems was performed to: 1) describe the epidemiology of the 2010 dengue epidemic, including disease severity; 2) determine the proportion of primary and secondary DENV infections, and the molecular epidemiology of the DENVs responsible for the epidemic; and 3) describe relationships between demographic variables (e.g. age, sex, municipality of residence) and characteristics of illness (e.g. infecting DENV-type, severity of illness). This investigation underwent institutional review at CDC and was determined to be public health practice and not research, including the post-hoc determinations of DENV molecular epidemiology and primary/secondary infection rates in reported cases; as such, Institutional Review Board approval was not required.
Surveillance data from five sources were used to identify cases. First, Centers for Disease Control and Prevention Dengue Branch (CDC-DB) and Puerto Rico Department of Health (PRDH) jointly operate the island-wide Passive Dengue Surveillance System (PDSS) that requires an acute serum specimen and completion of a Dengue Case Investigation Form (DCIF) (cdc.gov/dengue/resources/dengueCaseReports/DCIF_English.pdf)for case reporting and diagnostic testing. Second, the Enhanced Dengue Surveillance System (EDSS) operates solely in the municipalities of Patillas and Guayama and utilizes an on-site nurse epidemiologist to encourage case reporting and patient follow-up to obtain a convalescent serum specimen . Third, identification of fatal dengue cases is conducted via PDSS and EDSS , and enhanced fatal case surveillance was initiated in January 2010 in collaboration with the Instituto de Ciencias Forenses de Puerto Rico, which obtains blood and tissue specimens at autopsy from suspected dengue-related deaths. Fourth, PRDH operates the Notifiable Diseases Surveillance System (NDSS) wherein suspected dengue cases are reported without diagnostic testing at CDC-DB. Last, in addition to dengue diagnostic testing performed at CDC-DB for PDSS and EDSS, testing is performed by two private diagnostic laboratories outside of Puerto Rico according to their internal protocols . Diagnostic test results from these laboratories and patient data, including sex, age, and date of illness onset (if unavailable, specimen collection date was used instead), were entered into an independent database. Deduplication of individuals reported to more than one data source was achieved by matching records on name and date of birth and consolidation into a single case if two or more reports from any data source had symptom onset dates within 14 days of each other. As case-patients' travel history is not well captured via the surveillance systems used in this investigation, reported cases may represent both locally-acquired as well as travel-associated cases.
Dengue diagnostic testing
All diagnostic testing was performed at CDC-DB for serum specimens received through PDSS or EDSS using the following algorithm: acute specimens (collected ≤5 days after symptom onset) were tested by DENV-type-specific real-time reverse-transcriptase-polymerase chain reaction (RT-PCR)  adapted for high throughput using MDX-10 Universal and M48 systems (Qiagen, Valencia, CA); acute specimens collected 5 days after symptom onset and all convalescent specimens (collected ≥6 days after symptom onset) were tested for the presence of anti-DENV immunoglobulin M (IgM) antibody with an antibody-capture enzyme-linked immunosorbent assay (MAC ELISA) and a cut-off value of the OD450 of the specimen versus that of the negative control (ie. P/N ratio ) ≥2.0 , . All serum specimens from fatal cases were tested by both RT-PCR and MAC ELISA. Tissue specimens were tested at CDC Infectious Diseases Pathology Branch in Atlanta, GA by immunohistochemistry (IHC)  and flavivirus-specific RT-PCR  followed by sequencing.
A suspected dengue case was a dengue-like illness in a person in Puerto Rico whose health care provider: 1) submitted a DCIF and serum or tissue specimen to CDC-DB for dengue diagnostic testing; 2) submitted a serum specimen to a private laboratory for dengue diagnostic testing; or 3) reported the case via NDSS.
A laboratory-positive case was a suspected dengue case that met the following criteria for current (criteria 1 and 2) or recent (criterion 3) DENV infection: 1) detection of DENV nucleic acid in a serum or tissue specimen; 2) detection of DENV antigen in a tissue specimen; or 3) detection of anti-DENV IgM antibody in a serum specimen.
A laboratory-negative case was a suspected dengue case with: 1) no anti-DENV IgM antibody detected in a convalescent specimen; or 2) no DENV nucleic acid or antigen detected in a fatal case with only a tissue specimen submitted.
A laboratory-indeterminate case was a suspected dengue case with no DENV nucleic acid or anti-DENV IgM antibody detected in an acute specimen with no convalescent specimen available for testing.
Primary and secondary DENV infections
A representative sample of all RT-PCR-positive cases reported to PDSS or EDSS with illness onset between January 1 and December 31, 2010 was selected to determine the rates of primary and secondary DENV infection. Cases were stratified by age group with optimal allocation to allow for comparison between age groups, and further allocated to reflect the proportion of DENV-types that occurred during 2010 to allow for comparison between DENV-types and age groups. Sample size was calculated using an estimate of the proportion of secondary infections by age group based on data from the 2007 dengue epidemic , an error of 20%, 95% significance, and an expected 20% of specimens having insufficient specimen volume remaining for testing to be completed. Of the 1,000 selected cases, 818 had sufficient specimen volume and were tested at a dilution of 1:100 for the presence of anti-DENV IgG antibody by ELISA using DENV-1–4 antigen and a cut-off value of OD450≥0.15 , . A secondary DENV infection was defined by detection of anti-DENV IgG antibody in an acute specimen, and a primary DENV infection by lack of anti-DENV IgG antibody detection in an acute specimen.
Sequencing and phylogenetic analysis
Serum specimens with DENV-1 (n = 7), DENV-2 (n = 2) or DENV-4 (n = 4) detected by RT-PCR were randomly selected from municipalities with the highest incidence of the respective DENV-type and inoculated into cultured C6/36 cells; the presence of virus was confirmed by RT-PCR and indirect immunofluorescence . Isolates were further propagated and viral RNA was extracted from culture supernatants using the M48 BioRobot System (Qiagen; Valencia, CA). The envelope glycoprotein (E) gene was amplified and sequenced; sequence data were restricted to the E gene open reading frame (1,485 basepairs). Multiple sequence alignment was performed using MUSCLE available in MEGA 5 (megasoftware.net) and GTR+Γ+I4 was selected as the best nucleotide substitution model as determined by MODELTEST v3.7. Genetic relatedness was inferred and represented with phylogenetic trees using the maximum likelihood method in MEGA 5. MCMC was run in BEAST v1.6.1 (beast.bio.ed.ac.uk) under Bayesian skyline prior, constructed in TreeAnnotator found in the same BEAST package, and visualized in FigTree v1.3. Both trees rendered almost identical tree topologies, therefore confirming genetic relatedness. Evolutionary distances were corroborated by pairwise alignment in BioEdit v7.1.3 and E gene sequences from GenBank were included in the phylogenetic tree to support tree topology by currently circulating genotype. Tree topology was supported by bootstrapping with 1,000 replicates. Genotypes were referred to by previously described nomenclature , .
The frequencies of clinical, demographic and laboratory data were calculated by performing descriptive analyses of all suspected dengue cases identified in 2010. Rates of suspected dengue and laboratory-positive cases were calculated using population denominators obtained from the 2010 United States Census . Statistical differences in proportions were tested by applying the Chi-squared test and Fisher's exact test when applicable. Unless otherwise noted, relative risk ratios were used to calculate all differences between effect sizes. All data analyses were conducted using SAS 9.2 (SAS Institute Inc., Cary, NC), graphs were produced in Microsoft Excel (Microsoft Corp., Redmond, WA), and maps were created using ArcView (ESRI, Redlands, CA).
We identified 26,766 suspected dengue cases with illness onset between January 1 and December 31, 2010 (7.2 suspected dengue cases per 1,000 residents). Of these, 22,496 (84.0%) were reported to PDSS, 1,846 (6.9%) were identified though diagnostic testing at a private laboratory, 1,304 (4.9%) were reported to NDSS, and 1,120 (4.2%) were reported to EDSS (Fig. S1). Suspected dengue cases exceeded the PDSS epidemic threshold in the first week of 2010, increased steeply in week 20 (May 14–20), and peaked at 1,157 in week 32 (August 6–12) (Fig. 1). Suspected dengue cases slowly declined thereafter and returned to below the historic average in mid-December.
Surveillance data from cases reported via the Passive Dengue Surveillance System, Enhanced Dengue Surveillance System, Notifiable Diseases Surveillance System, or private laboratory dengue diagnostic test results were compiled and grouped by diagnostic test result as indicated.
Of all suspected dengue cases, 25,852 (96.6%) had a specimen tested for evidence of DENV infection, of which 25,246 (97.7%) were tested by CDC-DB and the remainder by a private laboratory; paired specimens were available for 1,996 (7.5%) cases. Of all cases with a specimen tested, 3,664 (14.2%) were laboratory-negative, 10,140 (39.2%) were laboratory-indeterminate, and 12,048 (46.6%) were laboratory-positive (3.2 laboratory-positive cases per 1,000 residents). The median weekly proportion of cases that tested laboratory-positive was 48.3%, and was highest (64.5%) in week 24 (June 11–17) and lowest (11.1%) in week 53 (December 31).
Laboratory-positive case-patients resided in all 78 municipalities of Puerto Rico (Fig. 2A), and the median rate of laboratory-positive cases by municipality was 2.68 per 1,000 residents. Rates were the highest in the municipality of Patillas (16.34 cases per 1,000 residents), the southeastern municipality where the EDSS site is located , and lowest in Aibonito (0.12 cases per 1,000 residents) in the mountainous center of Puerto Rico. Of 7,426 RT-PCR-positive cases, DENV-1 was detected in 5,126 (69.0%) and incidence was highest in the southeast (Fig. 2B). DENV-2 was detected in 545 (7.3%) cases primarily in the west (Fig. 2C), whereas DENV-4 was detected in 1,757 (23.7%) cases and incidence was highest in south-central and northwestern Puerto Rico (Fig. 2D). DENV-3 was detected in just two (<0.1%) cases in early 2010.
Rates were calculated by dividing case numbers by municipality-specific populations and grouping by quintile of rate of all laboratory-positive cases. Rates shown are: (A) All laboratory-positive cases; or laboratory-positive cases with DENV-1 (B), DENV-2 (C), or DENV-4 (D) detected by RT-PCR.
The age distribution of laboratory-positive cases was significantly different from suspected dengue cases only for case-patients between 30 and 69 years of age (Fisher's exact, p≤0.04). The median age of laboratory-positive case-patients was 18 years (Table 1). The most affected age group was 10–14 year olds (7.8 cases per 1,000 individuals), followed by 15–19 year olds (7.4 cases per 1,000 individuals) (Fig. 3A). Five-to-nine year olds were the next most affected age group followed by individuals <1 year of age (4.6 and 4.1 cases per 1,000 individuals, respectively). Individuals 50–59 years of age were the least affected age group (1.7 cases per 1,000 individuals).
A: Age distribution and rates of laboratory-positive cases; B: Age distribution and incidence of RT-PCR-positive cases by infecting DENV-type; C: Primary and secondary DENV infections by age group from a representative sample of RT-PCR-positive cases; error bars indicate standard error of the mean; denominators by age group are 15, 21, 73, 146, 162, 115, 74, 66, 54, 61 and 31, respectively.
The distribution of RT-PCR-positives cases among age groups was not significantly different from that of laboratory-positive cases (Fisher's exact, p>0.05) except for the 50–59 year-old age group, for which serum specimens were collected later (median: 6 days post-illness onset [DPO]) than all other age groups (median: 4 DPO) (Fisher's exact, p = 0.04) and thus tested less frequently by RT-PCR. Despite this, the distribution of DENV-types was not consistent among age groups (Fig. 3B). The strong majority (89.3%) of RT-PCR-positive cases in individuals 1–4 years of age were due to infection with DENV-1, whereas 8.1% and 2.6% were due to infection with DENV-4 and -2, respectively. The percent of infections due to DENV-1 decreased and those due to DENV-4 increased with age until a plateau of approximately 60% DENV-1, 30% DENV-4 and 10% DENV-2 was reached in the 20–29 year old age group.
Primary and secondary DENV infections
From the sample of 818 RT-PCR-positive specimens tested for primary versus secondary DENV infection, 169 (20.7%) were primary and 649 (79.3%) were secondary. The median age of individuals experiencing primary infection was 14 years, compared to 23 years for individuals experiencing secondary infection. Eighty-one percent of individuals 1–4 years of age had primary infection and were the only age group for which primary infection was significantly more common than secondary (p = 0.003) (Figure 3C). More than 89% of infections in all adult age groups (i.e. age ≥20 years) were secondary. The frequency with which anti-DENV IgG antibody was detected in specimens taken from infants was likely due to the presence of maternal antibody .
Whereas 28.5% of all DENV-1 infections were primary, significantly fewer DENV-2 (6.8%) and DENV-4 (7.1%) cases were primary infections (p<0.0001) (Table 2). Calculation of relative risk ratios (RR) indicated that individuals infected with DENV-1 were 4.2 and 4.0 times more likely to be experiencing primary infection than were individuals infected with DENV-2 or -4, respectively (Table 2).
Sequencing and phylogenetic analyses of randomly selected DENV isolates showed that DENV-1 belonged to the American-African genotype (genotype V ), but to a clade distinct from virus isolated during the 1998 Puerto Rico epidemic (Fig. 4A). Available sequence data suggest that close ascendants of the 2010 DENV-1 clade had been circulating in Puerto Rico and the Caribbean since at least 2006 (Fig. 4A). DENV-2 sequencing indicated that the virus belongs to clade 1B of the American-Asian genotype (genotype IIIb ) (Fig. 4B), which is composed of DENV strains endemic to Puerto Rico . DENV-4 belonged to the Indonesian genotype (genotype II ), but was distinct from virus isolated in 1998 (Fig. 4C). Viruses closely-related to the DENV-4 isolated in 2010 were first detected in Puerto Rico in 2004 (Fig. 4C).
Each phylogeny was tested with 1,000 bootstrapping cycles. Each taxa label consists of country of origin (PR = Puerto Rico), year of virus isolation, and GenBank accession number. Viruses isolated and sequenced for this investigation are labeled with a black dot. Genotype names were based on previously published phylogenies , . All outgroups have been removed. A: Phylogenies were constructed using 29 DENV-1 E gene sequences: seven from Puerto Rico in 2010, and 22 obtained from GenBank to represent the three main genotypes: American-African, South Pacific, and Asian. B: Phylogenies were constructed using 24 DENV-2 E gene sequences: two from Puerto Rico in 2010, and 22 obtained from GenBank to represent the three main genotypes: American-Asian, Cosmopolitan, and Asian II. C: Phylogenies were constructed using 26 DENV-4 E gene sequences: four from Puerto Rico in 2010, and 22 obtained from GenBank to represent the two main genotypes: Indonesian and South East Asian.
Of 12,048 laboratory-positive cases, 31.5% had at least one hemorrhagic manifestation and sufficient clinical data was provided to classify 74.0% as dengue and 2.4% as DHF (Table 1). Nearly half (49.7%) of all laboratory-positive cases had dengue with at least one warning sign, and 11.1% had severe dengue. Of 128 suspected dengue deaths, 40 (31.3%) were laboratory-positive cases. While adults represented nearly half of laboratory-positive cases with dengue (47.1%), dengue with warning signs (44.6%), and severe dengue (49.7%), they accounted for nearly all (92.5%) fatal dengue cases. Laboratory-positive severe and fatal dengue occurred at a rate of 0.36 and 0.01 cases per 1,000 residents, respectively; laboratory-positive fatal dengue cases occurred at a rate of 30.0 per 1,000 severe dengue cases. From the sample of cases for which primary and secondary DENV infection status was determined, secondary infection was identified in 102 (87.9%) case-patients with severe dengue and 547 (77.9%) case-patients without severe dengue (RR = 1.2; 95% CI = 1.1–1.2).
Case-patients with DHF were more likely to have been infected with DENV-4 than DENV-1, and those with severe dengue were more likely to have been infected with DENV-4 than DENV-1 or -2 (Table 2). There was no significant difference between infection with DENV-1, -2 or -4 and likelihood of being a fatal case.
In 2010, Puerto Rico experienced the largest and longest dengue epidemic ever documented on the island. In total, more than 12,000 individuals had laboratory-confirmed dengue, of which more than 1,300 experienced severe dengue and 40 died. The most common DENV identified was DENV-1, and 1–4 years old were the only age group more frequently experiencing a primary versus secondary DENV infection. Individuals infected with DENV-1 were four times more likely to have a primary infection than were those infected with DENV-2 or -4. A strength of this investigation was utilization of multiple surveillance systems to identify all reported suspect dengue cases. However, a minor weakness was that data obtained from each system may not be directly comparable due to different diagnostic algorithms used by CDC-DB and private laboratories, and we were not able to determine status of primary versus secondary infection or perform sequencing on specimens from private laboratories. Because private laboratories contributed <5% of all laboratory-positive dengue cases, this likely did not affect the conclusions of this investigation.
The 2010 dengue epidemic was similar in several respects to the 1998 epidemic: both began in January during El Niño events accompanied by above average temperatures, which while not a determinant of epidemics in Puerto Rico  may contribute to increased DENV transmission ; and both epidemics peaked in week 32 of the calendar year and were predominated by transmission of DENV-1 and -4 . A notable difference was that DENV-3 was essentially absent in 2010, whereas it accounted for ~6% of cases during the 1998 epidemic . DENV-3 was re-introduced into Puerto Rico in 1998 following a 20-year absence and was the predominant virus-type in the 2007 dengue epidemic . Thus, susceptibility to DENV-3 infection was likely high in 1998 and low in 2010, which likely explains these observations.
The American-African and Indonesian genotypes of DENV-1 and -4 have been circulating in Puerto Rico since introduced in 1978 and 1981, respectively , . However, the DENV-1 isolated in 2010 was distinct from the DENV-1 isolated during the 1998 epidemic (Fig. 4A and ) and was more closely related to the DENV-1 isolated during the 2007 epidemic (Fig. 4A). Similarly, the DENV-4 isolated during the 2010 epidemic was distinct from the DENV-4 isolated in 1998 and was more closely related to viruses circulating since 2004 (Fig. 4B). These findings suggest that DENV-1 and -4 may have both experienced clade replacements at some point after 1998 but prior to 2007. After the re-introduction of DENV-3 into Puerto Rico in 1998, DENV-1 was not detected between 2001 and 2006 and DENV-4 was not detected between 2000 and 2005 . Nonetheless, apparent re-introductions of DENV-1 in 2007 and DENV-4 in 2006 were soon followed by the disappearance of DENV-3 in 2010 (this paper and ). In place of the convenience sample used in this investigation to describe the DENVs responsible for the epidemic, sequencing of a representative sample of specimens and longitudinal sequence analysis will be necessary to both confirm apparent clade replacements and determine if other DENV clades contributed to the 2010 epidemic.
Similar to previous epidemics in Puerto Rico (Table S1), 10–19 year olds were most affected during the 2010 epidemic; however, unlike previous epidemics, 5–9 year olds were the next most affected age group. The median age of individuals experiencing secondary DENV infection declined from 27 years in 2007  to 23 years in 2010, likely due to the relative proximity of the periods of high infection pressure. Taken together, these observations indicate an increase in incidence of dengue and a decrease in the age of secondary infection, suggesting that the overall force of DENV transmission may have been higher in 2010 than in previous epidemic years.
The observation that DENV-2 and -4 cause relatively infrequent clinical apparent illness upon primary DENV infection is consistent with previous studies –. Similarly, our finding that DENV-1 was a more frequent cause of clinically apparent illness upon primary infection has also been previously reported , , including the observation of increased disease severity during primary infection with DENV-1 compared to other DENV-types , , . Nonetheless, of 545 DENV-2 and 1,755 DENV-4 infections, roughly 7% were primary, indicating that primary infection with these DENVs can cause clinically apparent illness, contrary to previous assertions , . The relative abundance of DENV-1 compared to DENV-2 and -4 is unlikely to be responsible for the observed differences in likelihood of causing clinically apparent illness upon primary infection, as relative risk ratios compare the proportion of exposed individuals experiencing the outcome of interest. This is supported by the findings in the 1–4 year-old age group, of which ~80% experienced a primary infection with DENV-1. Alternative explanations for these observations include potential variations in the sensitivity of detection of DENV-type-specific anti-DENV IgG antibody and differences in force of infection between the DENV-types circulating in 2010.
We also saw that DENV-1 and -2 were less frequently a cause of severe dengue than DENV-4. This is in contrast to previous studies where DENV-1 was a more frequent cause of DHF than DENV-4 , and a study where DENV-2 was twice as likely to result in DHF as DENV-4 . Possible explanations for these differences include: the comparatively small number of DENV-4 infections observed in previous studies; differences in clade and/or viral fitness leading to differential pathogenicity , , ; and/or the DENV-type(s) and sequence to which individuals were previously exposed, which may affect the likelihood of developing severe dengue , , .
This investigation had several limitations. First, because individuals experiencing secondary infection may have a diminished anti-DENV IgM antibody response , suspected dengue cases tested solely for anti-DENV IgM antibody may have been misclassified. Second, although DENV is the sole flavivirus known to cause clinically apparent illness in humans in Puerto Rico (CDC, unpublished data), some proportion of anti-DENV IgM or IgG positive results could have been due to infection with or vaccination against another flavivirus , resulting in misclassification. Third, because clinical data was provided for >90% of case-patients on only one occasion and some data variables were incompletely reported (e.g. only 56% of suspected cases had a reported status of hospitalization), severity of disease and the rates of dengue with warning signs and severe dengue reported here were likely underestimated. Finally, the description of the epidemiology and molecular characteristics of dengue reported here is only representative of reported, clinically apparent DENV infections and may not be reflective of asymptomatic and sub-clinical DENV infections.
The 2010 dengue epidemic in Puerto Rico demonstrated that dengue continues to be a public health concern for Puerto Rico residents and visitors, and surveillance systems and control initiatives should continue to be supported and strengthened. This epidemic also highlights the need for effective primary prevention tools such as a dengue vaccine to reduce disease morbidity and mortality.
Summary of epidemiologic data from previous dengue epidemics in Puerto Rico.
Flow diagram of data sources, diagnostic test results, and sub-analyses of suspected dengue cases, Puerto Rico, 2010. A: Data sources and diagnostic test results. B: Sub-analyses using RT-PCR-positive specimens. PDSS = Passive Dengue Surveillance System; PrivLab = private diagnostic laboratories; NDSS = National Disease Surveillance System; EDSS = Enhanced Dengue Surveillance System; IHC = immunohistochemistry; IgG ELISA = anti-DENV immunoglobulin G enzyme-linked immunosorbent assay; RT-PCR = real-time reverse-transcriptase polymerase chain reaction; DENV = dengue virus; * = includes two co-infections.
We thank the Instituto de Ciencias Forenses de Puerto Rico and CDC Infectious Diseases Pathology Branch for collection and diagnostic testing of autopsy specimens, respectively. We also thank Oscar Padró for maintenance of all CDC-DB databases, Candimar Colón and Manuela Beltrán for management of CDC-DB molecular and serologic diagnostics laboratories, respectively, and Michael Johansson for helpful discussions and manuscript review. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.
Conceived and designed the experiments: TMS EH GAS JLMJ LMS HSM KMT. Performed the experiments: TMS EH GAS JLMJ LMS. Analyzed the data: TMS EH GAS JLMJ LMS HSM KMT. Contributed reagents/materials/analysis tools: AR RLRA LGF. Wrote the paper: TMS GAS HSM KMT.
- 1. Gubler DJ (2002) Epidemic dengue/dengue hemorrhagic fever as a public health, social and economic problem in the 21st century. Trends Microbiol 10: 100–103. doi: 10.1016/s0966-842x(01)02288-0
- 2. World Health Organization (2009) Dengue: Guidelines for Diagnosis, Treatment, Prevention and Control. First edition. Geneva: WHO. http://whqlibdoc.who.int/publications/2009/9789241547871_eng.pdf.
- 3. World Health Organization (1997) Dengue haemorrhagic fever: diagnosis, treatment, prevention and control. Second edition. Geneva: WHO. http://www.who.int/csr/resources/publications/dengue/Denguepublication/en/.
- 4. Effler PV, Pang L, Kitsutani P, Vorndam V, Nakata M, et al. (2005) Dengue fever, Hawaii, 2001–2002. Emerg Infect Dis 11: 742–749. doi: 10.3201/eid1105.1063
- 5. Rodhain R, Rosen L., editor (1997) Dengue and dengue hemorrhagic fever. New York: CAB International. 45–60 p.
- 6. Murphy BR, Whitehead SS (2011) Immune response to dengue virus and prospects for a vaccine. Annu Rev Immunol 29: 587–619. doi: 10.1146/annurev-immunol-031210-101315
- 7. Halstead SB, Nimmannitya S, Cohen SN (1970) Observations related to pathogenesis of dengue hemorrhagic fever. IV. Relation of disease severity to antibody response and virus recovered. Yale J Biol Med 42: 311–328.
- 8. Whitehead SS, Blaney JE, Durbin AP, Murphy BR (2007) Prospects for a dengue virus vaccine. Nat Rev Microbiol 5: 518–528. doi: 10.1038/nrmicro1690
- 9. United States Census Bureau (2010) American Fact Finder. http://factfinder2.census.gov/faces/nav/jsf/pages/index.xhtml.
- 10. King W (1917) The epidemic of dengue in Puerto Rico: 1915. New Orleans Medical Surgergy 69: 573–579.
- 11. Neff JM, Morris L, Gonzalez-Alcover R, Coleman PH, Lyss SB, et al. (1967) Dengue fever in a Puerto Rican community. Am J Epidemiol 86: 162–184.
- 12. Likosky WH, Calisher CH, Michelson AL, Correa-Coronas R, Henderson BE, et al. (1973) An epidermiologic study of dengue type 2 in Puerto Rico, 1969. Am J Epidemiol 97: 264–275.
- 13. Lopez-Correa RH, Cline BL, Ramirez-Ronda C, Bermudez R, Sather GE, et al. (1978) Dengue fever with hemorrhagic manifestations: a report of three cases from Puerto Rico. Am J Trop Med Hyg 27: 1216–1224.
- 14. Morens DM, Rigau-Perez JG, Lopez-Correa RH, Moore CG, Ruiz-Tiben EE, et al. (1986) Dengue in Puerto Rico, 1977: public health response to characterize and control an epidemic of multiple serotypes. Am J Trop Med Hyg 35: 197–211.
- 15. Gubler DJ (2006) Dengue/dengue haemorrhagic fever: history and current status. Novartis Found Symp 277: 3–16; discussion 16–22, 71–13, 251–253 . doi: 10.1002/0470058005.ch2
- 16. Gubler DJ (1987) Dengue and dengue hemorrhagic fever in the Americas. P R Health Sci J 6: 107–111. doi: 10.1590/s1020-49892005000400001
- 17. Dietz V, Gubler DJ, Ortiz S, Kuno G, Casta-Velez A, et al. (1996) The 1986 dengue and dengue hemorrhagic fever epidemic in Puerto Rico: epidemiologic and clinical observations. P R Health Sci J 15: 201–210.
- 18. Rigau-Perez JG, Vorndam AV, Clark GG (2001) The dengue and dengue hemorrhagic fever epidemic in Puerto Rico, 1994–1995. Am J Trop Med Hyg 64: 67–74.
- 19. Rigau-Perez JG, Ayala-Lopez A, Garcia-Rivera EJ, Hudson SM, Vorndam V, et al. (2002) The reappearance of dengue-3 and a subsequent dengue-4 and dengue-1 epidemic in Puerto Rico in 1998. Am J Trop Med Hyg 67: 355–362.
- 20. Tomashek KM, Rivera A, Munoz-Jordan JL, Hunsperger E, Santiago L, et al. (2009) Description of a large island-wide outbreak of dengue in Puerto Rico, 2007. Am J Trop Med Hyg 81: 467–474.
- 21. Ramos MM, Arguello DF, Luxemburger C, Quinones L, Munoz JL, et al. (2008) Epidemiological and clinical observations on patients with dengue in Puerto Rico: results from the first year of enhanced surveillance–June 2005–May 2006. Am J Trop Med Hyg 79: 123–127.
- 22. Tomashek KM, Gregory CJ, Rivera Sanchez A, Bartek MA, Garcia Rivera EJ, et al. (2012) Dengue deaths in Puerto Rico: lessons learned from the 2007 epidemic. PLoS neglected tropical diseases 6: e1614. doi: 10.1371/journal.pntd.0001614
- 23. Prince HE, Matud JL, Lieberman JM (2011) Dengue virus immunoglobulin M detection in a reference laboratory setting during the 2010 dengue virus outbreak on Caribbean islands. Clin Vaccine Immunol 18: 1104–1107. doi: 10.1128/cvi.05096-11
- 24. Johnson BW, Russell BJ, Lanciotti RS (2005) Serotype-specific detection of dengue viruses in a fourplex real-time reverse transcriptase PCR assay. J Clin Microbiol 43: 4977–4983. doi: 10.1128/jcm.43.10.4977-4983.2005
- 25. Burke DS, Nisalak A, Ussery MA (1982) Antibody capture immunoassay detection of japanese encephalitis virus immunoglobulin m and g antibodies in cerebrospinal fluid. J Clin Microbiol 16: 1034–1042.
- 26. Martin DA, Biggerstaff BJ, Allen B, Johnson AJ, Lanciotti RS, et al. (2002) Use of immunoglobulin m cross-reactions in differential diagnosis of human flaviviral encephalitis infections in the United States. Clin Diagn Lab Immunol 9: 544–549. doi: 10.1128/cdli.9.3.544-549.2002
- 27. Guarner J, Bhatnagar J, Shieh WJ, Nolte KB, Klein D, et al. (2007) Histopathologic, immunohistochemical, and polymerase chain reaction assays in the study of cases with fatal sporadic myocarditis. Hum Pathol 38: 1412–1419.
- 28. Bhatnagar J, Blau DM, Shieh WJ, Paddock CD, Drew C, et al. (2012) Molecular detection and typing of dengue viruses from archived tissues of fatal cases by rt-PCR and sequencing: diagnostic and epidemiologic implications. The American journal of tropical medicine and hygiene 86: 335–340. doi: 10.4269/ajtmh.2012.11-0346
- 29. Johnson AJ, Martin DA, Karabatsos N, Roehrig JT (2000) Detection of anti-arboviral immunoglobulin G by using a monoclonal antibody-based capture enzyme-linked immunosorbent assay. J Clin Microbiol 38: 1827–1831.
- 30. Miagostovich MP, Nogueira RM, dos Santos FB, Schatzmayr HG, Araujo ES, et al. (1999) Evaluation of an IgG enzyme-linked immunosorbent assay for dengue diagnosis. J Clin Virol 14: 183–189. doi: 10.1016/s1386-6532(99)00059-1
- 31. Kuno G, Gubler DJ, Velez M, Oliver A (1985) Comparative sensitivity of three mosquito cell lines for isolation of dengue viruses. Bull World Health Organ 63: 279–286.
- 32. Twiddy SS, Farrar JJ, Vinh Chau N, Wills B, Gould EA, et al. (2002) Phylogenetic relationships and differential selection pressures among genotypes of dengue-2 virus. Virology 298: 63–72. doi: 10.1006/viro.2002.1447
- 33. Rico-Hesse R (2003) Microevolution and virulence of dengue viruses. Adv Virus Res 59: 315–341. doi: 10.1016/s0065-3527(03)59009-1
- 34. Goncalvez AP, Escalante AA, Pujol FH, Ludert JE, Tovar D, et al. (2002) Diversity and evolution of the envelope gene of dengue virus type 1. Virology 303: 110–119. doi: 10.1006/viro.2002.1686
- 35. Bennett SN, Holmes EC, Chirivella M, Rodriguez DM, Beltran M, et al. (2006) Molecular evolution of dengue 2 virus in Puerto Rico: positive selection in the viral envelope accompanies clade reintroduction. J Gen Virol 87: 885–893. doi: 10.1099/vir.0.81309-0
- 36. McElroy KL, Santiago GA, Lennon NJ, Birren BW, Henn MR, et al. (2011) Endurance, refuge, and reemergence of dengue virus type 2, Puerto Rico, 1986–2007. Emerg Infect Dis 17: 64–71. doi: 10.3201/eid1701.100961
- 37. Lanciotti RS, Gubler DJ, Trent DW (1997) Molecular evolution and phylogeny of dengue-4 viruses. J Gen Virol 78 (Pt 9) 2279–2284.
- 38. Johansson MA, Cummings DA, Glass GE (2009) Multiyear climate variability and dengue–El Nino southern oscillation, weather, and dengue incidence in Puerto Rico, Mexico, and Thailand: a longitudinal data analysis. PLoS Med 6: e1000168. doi: 10.1371/journal.pmed.1000168
- 39. Johansson MA, Dominici F, Glass GE (2009) Local and global effects of climate on dengue transmission in Puerto Rico. PLoS neglected tropical diseases 3: e382. doi: 10.1371/journal.pntd.0000382
- 40. Gubler DJ, Trent DW (1993) Emergence of epidemic dengue/dengue hemorrhagic fever as a public health problem in the Americas. Infect Agents Dis 2: 383–393. doi: 10.3201/eid0102.950204
- 41. Anez G, Heisey DA, Espina LM, Stramer SL, Rios M (2012) Phylogenetic analysis of dengue virus types 1 and 4 circulating in Puerto Rico and Key West, Florida, during 2010 Epidemics. Am J Trop Med Hyg 87: 548–553. doi: 10.4269/ajtmh.2012.12-0091
- 42. Santiago GA, McElroy-Horne K, Lennon NJ, Santiago LM, Birren BW, et al. (2012) Reemergence and decline of dengue virus serotype 3 in Puerto Rico. J Infect Dis 206: 893–901. doi: 10.1093/infdis/jis426
- 43. Fried JR, Gibbons RV, Kalayanarooj S, Thomas SJ, Srikiatkhachorn A, et al. (2010) Serotype-specific differences in the risk of dengue hemorrhagic fever: an analysis of data collected in Bangkok, Thailand from 1994 to 2006. PLoS Negl Trop Dis 4: e617. doi: 10.1371/journal.pntd.0000617
- 44. Nisalak A, Endy TP, Nimmannitya S, Kalayanarooj S, Thisayakorn U, et al. (2003) Serotype-specific dengue virus circulation and dengue disease in Bangkok, Thailand from 1973 to 1999. Am J Trop Med Hyg 68: 191–202.
- 45. Buchy P, Vo VL, Bui KT, Trinh TX, Glaziou P, et al. (2005) Secondary dengue virus type 4 infections in Vietnam. Southeast Asian J Trop Med Public Health 36: 178–185.
- 46. Guzman MG, Kouri G, Valdes L, Bravo J, Alvarez M, et al. (2000) Epidemiologic studies on Dengue in Santiago de Cuba, 1997. Am J Epidemiol 152: 793–799 discussion 804. doi: 10.1093/aje/152.9.793
- 47. Vaughn DW, Green S, Kalayanarooj S, Innis BL, Nimmannitya S, et al. (2000) Dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity. J Infect Dis 181: 2–9. doi: 10.1086/315215
- 48. Anantapreecha S, Chanama S, A An, Naemkhunthot S, Sa-Ngasang A, et al. (2005) Serological and virological features of dengue fever and dengue haemorrhagic fever in Thailand from 1999 to 2002. Epidemiol Infect 133: 503–507. doi: 10.1017/s0950268804003541
- 49. Thu HM, Lowry K, Myint TT, Shwe TN, Han AM, et al. (2004) Myanmar dengue outbreak associated with displacement of serotypes 2, 3, and 4 by dengue 1. Emerg Infect Dis 10: 593–597. doi: 10.3201/eid1004.030216
- 50. Balmaseda A, Hammond SN, Perez L, Tellez Y, Saborio SI, et al. (2006) Serotype-specific differences in clinical manifestations of dengue. Am J Trop Med Hyg 74: 449–456.
- 51. Nishiura H, Halstead SB (2007) Natural history of dengue virus (DENV)-1 and DENV-4 infections: reanalysis of classic studies. J Infect Dis 195: 1007–1013. doi: 10.1086/511825
- 52. Fagbami AH, Mataika JU, Shrestha M, Gubler DJ (1995) Dengue type 1 epidemic with haemorrhagic manifestations in Fiji, 1989–90. Bull World Health Organ 73: 291–297.
- 53. Rico-Hesse R (2007) Dengue virus evolution and virulence models. Clin Infect Dis 44: 1462–1466. doi: 10.1086/517587
- 54. Ohainle M, Balmaseda A, Macalalad AR, Tellez Y, Zody MC, et al. (2011) Dynamics of dengue disease severity determined by the interplay between viral genetics and serotype-specific immunity. Sci Transl Med 3: 114ra128. doi: 10.1126/scitranslmed.3003084
- 55. Endy TP, Nisalak A, Chunsuttitwat S, Vaughn DW, Green S, et al. (2004) Relationship of preexisting dengue virus (DV) neutralizing antibody levels to viremia and severity of disease in a prospective cohort study of DV infection in Thailand. J Infect Dis 189: 990–1000. doi: 10.1086/382280
- 56. Kyle JL, Harris E (2008) Global spread and persistence of dengue. Annu Rev Microbiol 62: 71–92. doi: 10.1146/annurev.micro.62.081307.163005
- 57. Gubler DJaK, G. (1997) Dengue and Dengue Hemorrhagic Fever: CABI. 478 p.
- 58. Calisher CH, Karabatsos N, Dalrymple JM, Shope RE, Porterfield JS, et al. (1989) Antigenic relationships between flaviviruses as determined by cross-neutralization tests with polyclonal antisera. J Gen Virol 70 (Pt 1) 37–43. doi: 10.1099/0022-1317-70-1-37