Evaluation of an influenza-like illness case definition in the diagnosis of influenza among patients with acute febrile illness in cambodia

In December 2006, a clinic and hospital-based acute febrile illness surveillance was implemented at nine Cambodian government medical clinics. Five of these sites were located in Operational District A (peri-urban) and four were in Operational District B (rural). All participating field sites were within 50 kilometers of Phnom Penh in south-central Cambodia. Patients were recruited by study site staff if they had a recorded temperature ≥ 38.0°C lasting at least 24 hours but not greater than 10 days, were two years of age or older, and, after medical examination, had no obvious source of infection. A healthcare provider in each clinic obtained written informed consent, administered a pre-tested enrollment questionnaire, performed a medical examination and collected clinical specimens per study protocol[19]. Influenza-like illness was defined according the WHO guidelines, which included, documented fever (≥ 38.0°C) and cough or sore throat. For this study, only patients enrolled I the surveillance for acute febrile illness from July 2008 through December 2008 were included for analysis; corresponding to influenza season in Cambodia.

For each enrolled patient, one throat and one nasal swab were collected. For nasal swabs, a dry polyester swab was inserted into the nostril parallel to the palate, slowly withdrawn, and placed in a vial containing 2 - 3 milliliters of virus transport medium (VTM). For throat swabs, both tonsils and the posterior pharynx were swabbed vigorously, and the swab placed in 2 - 3 milliliters of VTM. All inoculated vials were kept at 4°C until transported between 24 and 72 hours after collection to the Naval Medical Research Unit No. 2 (NAMRU-2) located at the campus of the Cambodian National Institute of Public Health (NIPH).

Ribonucleic acid (RNA) was extracted from nasal and throat swabs using QIAamp viral RNA mini kits (QIAGEN, Hilden, Germany) following the manufacturer's instructions and stored at -70°C. The influenza virus genome was detected using a reverse real-time PCR (rRT-PCR) assay developed to detect influenza A/H1, A/H3, A/H5, and B virus subtypes. Real-time assays were developed at the Centers for Disease Control and Prevention (Atlanta, GA, USA). Real time assays and sequence information are available upon request from Dr. Steve Lindstrom under the terms of a material transfer agreement. One-step rRT-PCR was performed in a final volume of 25 μl containing 5 μl of extracted RNA, 12.5 μl of buffer mix and 0.5 μl Superscript III/Platinum Taq-Enzyme mix, 20 units of RNAse-out (Invitrogen, Carlsbad, CA, USA), 0.8 μM for each primer and 0.2 μM of probe. The Rotor-Gene 6000 real-time thermocyler (Corbett Life Science, Sydney, Australia) was used for all PCR reactions. The thermocycling parameters for all targets consisted of 50°C for 30 minutes, 95°C for 2 minutes, and 45 cycles with 95°C for 15 seconds, 55°C for 30 seconds (US CDC, Atlanta, GA, USA).

All data was double-data entered into MS Access (Microsoft Inc., Redmond, WA, USA). Data was imported into SAS v9.1 (SAS, Cary, NC), which was used for all statistical analyses. The definition of ILI (fever and cough or sore throat) was analyzed to determine sensitivity, specificity, positive predictive valve (PPV) and negative predictive value (NPV) comparing patients with and without laboratory evidence of influenza virus. Sensitivity was defined as the probability of having the ILI given laboratory-confirmed influenza; specificity was defined as the probability of not having the ILI when the patient does not have laboratory-confirmed influenza. The PPV was the probability of having laboratory-confirmed influenza when ILI was present; NPV is the probability of not having laboratory-confirmed influenza when ILI was not present[15]. Categorical data was analyzed by the use of either Chi-Square (expected cell frequency > 5) or Fisher's Exact Test (expected cell frequency ≤5). Continuous data were assessed for normality and if normally distributed, parametric statistics were used. If the data was non-normally distributed, then non-parametric testing was used. All statistical tests were two-tailed and significance was defined as p < 0.05.

Eligible subjects were voluntarily enrolled in accordance with an Institutional Review Board protocol approved by U.S. NAMRU-2 and the National Ethics Committee of the Royal Kingdom of Cambodia, Ministry of Health.