248 KASPER AND OTHERS kits (QIAGEN, Hilden, Germany) following manufacturer’s instruction and stored at 80 C. Influenza virus genome was detected using a standardized real-time reverse transcription (rRT)-PCR assay developed by the U.S. CDC to detect influenza A and B viruses as well as influenza A viruses of H1, H3, and H5 subtypes. Real time assays were developed at the CDC and assays and primer sequences are available upon request under a Material Transfer Agreement. One-step rRT-PCR was performed in a final volume of 25 mL containing 5 mL of extracted RNA, 12.5 mL of buffer mix, and 0.5 mL Superscript III/Platinum Taq-Enzyme mix, 20 U of RNAse- out (Invitrogen, Carlsbad, CA), 0.8 mM for each primer, and 0.2 mM 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 (U.S. CDC). In August 2009, a real-time PCR assay distributed by the U.S. CDC was introduced for the detection of pandemic H1N1 influenza virus. The Applied Biosystems 7500 real time thermocycler (Applied Biosystems, Foster City, CA) was used for all pH1N1 PCR reactions. Nested conventional PCR for dengue. Dengue virus genome and serotype detection was based on previously published procedures with modifications to the Dengue-1 primer.17,18 Nested reverse real-time PCR for chikungunya virus. The RNA was extracted from serum using QIAamp viral RNA mini kits (QIAGEN) following manufacturer’s instruction and stored at 80 C. Chikungunya virus genome was detected using a nested RT-PCR assay, which comprises two assay steps; RT-PCR and nested PCR (nPCR). The RT reactions were performed in 25 mL containing 5 mL of Access AMV-Tfl buffer (Promega, Madison WI), 1.5 mL of 25 mM MgSO4, 1 mL of 10 mM deoxynucleoside triphosphate (dNTP; final concen- tration, 200 mM), 20 pico moles each of all outer primers sets, 0.5 mL of AMV-reverse transcriptase, and 0.5 mL of 5 U Tfl polymerase (Promega), and 5 mL of RNA template. The PCR thermal cycling was performed as follows: 48 C for 45 minutes, 94 C for 2 minutes, and 40 cycles with 94 C for 1 minute, 60 C for 1 minute, 68 C for 1 minute, and then 68 C for 7 minutes. All thermal cycling was performed with Applied Biosystems type 9700 machine. Inner primer pairs are then used in separate nPCR reactions using GeneAmp PCR Core reagent (Applied Biosystems). The nPCR reactions were performed in 25 mL containing 2.5 mL of ABI PCR buffer, 1.5 mL of 25 mM MgCl2, 2.0 mL of 10 mM dNTP (final concentration 200 mM), 1.0 mL of 10 mM forward primer, and 1.0 mL of reverse primer, 0.25 mL of 5 units of AmpliTaq DNA Polymerase, and 5 mL of diluted (1:50) RT-PCR product. The cycle conditions of the nPCR reaction were as follows; 94 C for 2 minutes, and 40 cycles with 94 C for 30 seconds, 60 C for 30 seconds, 72 C for 30 seconds, and then 72 C for 10 minutes. Outer and inner primer pairs are designed to amplify three structural gene regions, Capsid (C), Envelope-2 (E2), and an area overlapping the 6K and E1 genes (6K/E1).14 The PCR products were visualized on 2% agarose gels stained with ethidium bromide under UV light. A positive result was indicated upon observation of a 200 base pair PCR product, representing the capsid, and at least one of the envelope targets is present. Quantitative real-time PCR (qPCR) for rickettsiae and Orientia tsutsugamushi (scrub typhus). Deoxyribonucleic acid (DNA) was extracted from serum using QIAamp DNA mini kits (QIAGEN) following manufacturer’s instruction and stored at 80 C. The qPCR assays developed to detect Rickett- sia spp. (Rick17), tick-borne rickettsiae (Trick), Rickettsia typhi (Rtyph), Rickettsia felis (Rfelis), and O. tsutsugamushi (Otsu47) were performed as previously described.19,20 Briefly, primer pairs were used in separate qPCR assays in a 25 mL final volume: Rick17, Trick, and Rtyph assays contained 2 mL of DNA, 12.5 mL of 2 +TaqMan Universal PCR Master Mix (Cat. no. ABI 4304437), 0.4 mM for each primer, and 0.3 mM of probe. Orientia tsutsugamushi qPCR assay contained 5 mL FIGURE 2. Catchment area (gray) of febrile surveillance study.