An enhanced surveillance system for AFI was established at the Saint Luke's Episcopal Hospital, in Ponce, Puerto Rico. Located ∼45 miles southwest of San Juan, Ponce is one of the 78 administrative municipalities of Puerto Rico and the second largest city (Figure 1). This hospital is a tertiary, acute-care facility that provides healthcare to patients from over 20 municipalities, has more than 54,000 patient visits to its ED annually,15 and is the main teaching affiliate of the Ponce School of Medicine.
Study population.
Patients of all ages were eligible to be enrolled if they met the following case definition of AFI: documented fever of 38.0°C or higher at presentation to the ED or history of fever that had persisted for 2–7 days without an identified source. Patients were excluded if after the initial physical examination by hospital physicians they had an identifiable source of fever including, but not limited to diagnoses of otitis media, sinusitis, bronchitis, pneumonia, cellulitis, impetigo, wound infection, urinary tract infection, osteomyelitis, or varicella.
Study protocol.
From September 29, 2009 through December 18, 2009, patients seeking medical care in the ED of St Luke's Episcopal Hospital who met the case definition for AFI were enrolled in the enhanced surveillance system (Figure 2). This surveillance included all elements of the PDSS established for decades and the addition of on-site CDC staff to help identify and collect data from AFI patients, including completion of the dengue case investigation form (DCIF), which accompanied specimens submitted to CDC's Dengue Branch for diagnostic testing. After physical examination, study personnel explained the purpose of the surveillance project and obtained verbal consent for participation. Participants were interviewed to collect DCIF data that included demographic, medical and clinical information, signs and symptoms at onset, and specimen collection dates. A tourniquet test was performed and results recorded by trained study personnel using standardized techniques as previously described.16 Two nasopharyngeal samples were collected to perform rapid antigen and reverse transcriptase-polymerase chain reaction (RT-PCR) testing for influenza. Attending physicians and patients were informed immediately of the results of the rapid influenza test that was performed on-site. One blood sample was collected for dengue diagnostic testing. Additional laboratory tests such as a complete blood count and urinalysis were performed at the discretion of the attending physician in the course of routine patient care but were not part of the study. If available, laboratory results, including white blood cell (WBC) count, platelet count, hematocrit, and albumin, were recorded on the DCIF. This protocol was reviewed and approved by the Human Subjects Institutional Review Board of the CDC and the Institutional Review Board of Ponce School of Medicine.
Laboratory testing.
Two nasopharyngeal samples were obtained at the same time. The first was tested on-site with the QuickVue Influenza A + B rapid influenza test (Quidel Corporation, San Diego, CA), and the second sample was placed in viral transport medium and refrigerated until transported to the CDC's Dengue Branch, San Juan, PR, for influenza testing by the CDC RT-PCR assay.17 As per PDSS protocol, 5 to 10 mL of venous blood was collected, immediately refrigerated at 4°C, centrifuged on-site to separate serum, and transported on ice within 3 days to the CDC's Dengue Branch for further testing. Serum samples were initially tested for DENV by serotype-specific RT-PCR,18 dengue-specific non-structural protein-1 (NS1), and by DENV-specific immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (anti-DENV MAC-ELISA).19 Serum samples from patients with severe disease (i.e., hospital admission or clinical signs including plasma leakage, fluid accumulation, respiratory distress, bleeding, and organ impairment) and from those who were influenza PCR-negative were subsequently transported on ice to CDC's Bacterial Zoonosis Branch in Atlanta for Leptospira testing. Specimens were screened for IgM antibodies to Leptospira by using the rapid dipstick ELISA ImmunoDOT kit (GenBio, Inc., San Diego, CA). Specimens with positive or borderline results with the ImmunoDOT kit were further tested by using the microscopic agglutination test (MAT).20 Patient sera were serially diluted in the MAT and mixed with a panel of 20 Leptospira reference antigens representing 17 serogroups. Resulting agglutination titers were read by using dark field microscopy, and the final titer was expressed as the reciprocal of the last well that agglutinated 50% of the antigen. Samples drawn within the first 3 days from symptom onset and with sufficient serum volume were shipped frozen to the Picornavirus Laboratory at CDC, Atlanta, and tested for enteroviruses by a pan-enterovirus (EV) RT-PCR that targets the conserved 5′ non-translated region of the genome.21
Definitions.
Acute febrile illness was defined as a patient with fever of 38°C or higher at presentation to ED or history of fever that persisted for 2–7 days with no localizing source.
Laboratory-positive dengue case: a patient with one or more of the following: 1) DENV RNA detected in serum by RT-PCR, 2) negative to positive anti-DENV IgM seroconversion in paired serum specimens, 3) a single positive anti-DENV IgM result in an acute-phase or convalescent-phase specimen (positive/negative antibody ratio ≥ 2.0),22 or 4) a positive DENV antigen detection by NS1 rapid test.
Laboratory-negative dengue case: a patient who does not meet criteria for a laboratory-positive dengue case and has no anti-DENV IgM detected in a serum specimen collected 6 or more days after onset of fever.
Laboratory-indeterminate dengue case: a patient with no DENV RNA, NS1 antigen or dengue virus or anti-dengue IgM antibodies detected in the acute sample submitted for diagnostic testing and no convalescent sample submitted for diagnostic testing.
Dengue fever, dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS) were defined by using the World Health Organization (WHO) criteria.23–27
Warning signs for severe dengue were defined as those that occurred within 48 hours of defervescence (about 3 to 7 days after symptoms onset) and included: severe abdominal pain or tenderness, persistent vomiting, clinical fluid accumulation, hemorrhagic manifestation, lethargy or restlessness, and liver enlargement.28
Laboratory-positive influenza case was defined as a patient with laboratory-confirmed infection detected by the CDC RT-PCR assay.17
Laboratory-positive leptospirosis case was defined by the following: 1) having positive IgM antibodies to Leptospira with the rapid dipstick ELISA ImmunoDOT kit and 2) a positive MAT for a single titer of > 1:400 or a fourfold rise in titer between acute and convalescent phase samples.20
Laboratory-positive EV case was defined as a patient with positive EV real-time RT-PCR assay and genotype identification by EV VP1 semi-nested RT-PCR and amplicon sequencing.21,29,30
Anemia was defined as a deficiency in the total number of erythrocytes in blood. A patient was considered to be anemic if its hemoglobin was less than the 2.5 percentile for age and sex.23,24
The WBC counts were classified as leucopenia (total WBC < 5,000/mm3) or leukocytosis (WBC > 11,000/mm3).31
Hemoconcentration was defined as an increase in hematocrit ≥ 20% above average for age or a decrease in hematocrit ≥ 20% of baseline following fluid replacement therapy.22
Analyses.
Data were entered into a Microsoft Access database (Microsoft Access 2007; Microsoft, Redmond, WA) and exported to SAS version 9.2 (SAS Institute, Cary, NC) for analysis. Outcome groups were identified as having confirmed dengue, confirmed influenza, confirmed leptospirosis, confirmed EV infection, or other AFI. Categorical variables were compared by using the χ2 test or Fisher's exact test as appropriate and continuous variables were compared by using the Student's t test and the Mann-Whitney U test (Wilcoxon rank-sum test) where applicable. Results with values P ≤ 0.05 were considered statistically significant.
Results
A total of 284 patients were enrolled (Table 1), and 172 (61%) met the case definition for influenza, dengue, leptospirosis, or enteroviral disease. Influenza A was identified in 138 (49%) patients, and all except two had a positive RT-PCR for pandemic 2009 influenza A H1N1 virus. No influenza B was identified. Dengue virus was identified in 32 (11%) patients; one of these patients had a dual infection with DENV-2 and influenza A H1N1 virus. Among patients laboratory positive for dengue, 20 were DENV RNA positive; 18 had DENV-4, 1 had DENV-1, and 1 had DENV-2. DENV-3 was not found. The remaining dengue patients were NS1 antigen positive (7 [23%]) or had a single positive anti-DENV IgM positive (4, 13%). Leptospira was detected in one patient; however, this patient also had influenza A H1N1 detected by RT-PCR. Enterovirus was detected in three patients and the genotype was determined in 2 patients (coxsackievirus A2 and coxsackievirus B4). Blood cultures, urine cultures, or both were performed in only 10 of all enrolled patients. Two patients had a positive urine culture; one positive for Escherichia coli and one for Staphylococcus saprophyticus. The two patients with dual diagnoses were excluded from the rest of the analysis.Influenza cases were more likely to be detected earlier in the study period than laboratory-positive dengue cases; most influenza cases were detected from October 1 through November 18, 2009, versus November 5 through December 16, 2009, for dengue (Figure 3). The three EV cases occurred during October. Overall, the patients ranged in age from 6 months to 82 years with a median age of 17.9 years (Table 2). Median age for patients with other AFI was 15.4 years compared with 20.3 years