M. weeks for growth. Serology is more convenient and sensitive than culture, but results are often delayed, and false-negative test results often occur early in the course of illness. Although not standardized, nucleic acid-based tests, such as PCR, provide fast and sensitive results. While such tests are not always available on site in many medical centers, the 24- to 48-h delay in transit time may still be acceptable given the higher diagnostic yield of PCR. Despite the obvious limitations of culture, physicians continue to order this test frequently. In recent years, ARUP Laboratories has received large numbers of requests, including more than 3,000/year for and more than 1,500/year for culture. Studies focusing on the value of culture either have been small in scale or have used type strains or patient isolates rather than direct patient specimens (8, 13). An accurate and reliable diagnosis of and is important to differentiate them from other common respiratory pathogens because their treatments differ (1). Being aware of the poor sensitivity of culture for these pathogens, we found the high numbers of test requests for and culture from respiratory tract specimens to be concerning and to require further investigation. To examine more closely the utility of culture for diagnosing respiratory syndromes, we compared its performance to those of nucleic acid testing and serology for detection of and and were retrospectively reviewed with a specific focus on respiratory specimens (e.g., nasal wash, nasopharyngeal swab, bronchoalveolar lavage, tracheal aspirate, sputum, and pleural fluid) for PCR and culture. Respiratory specimens were transported either refrigerated Ace2 or frozen, except for culture, for which only refrigerated specimens were transported. Additional data were collected for culture (1995 to 2003). enzyme-linked immunosorbent assay (ELISA) was performed only in 2005 to 2008, while microimmunofluorescence (MIF) was performed from 2003 to 2008. For serologic tests, data from IgM testing were collected since paired serology for IgG was rarely ordered. Subset analyses were performed for those specimens that were tested by both culture and another method. In 2008, culture-negative specimens for and were prospectively collected for PCR testing. The study protocol was approved by the University of Utah Institutional Review Board. For culture, respiratory specimens were diluted if viscous, vortexed, supplemented with amphotericin B and penicillin, and inoculated into SP-4 medium. The medium was observed daily for 21 days for a decrease in pH (a red to yellow color change). Positive cultures were confirmed by fluorescent antibody testing (Chemicon MA88285, Temecula, CA) or PCR. For and PCRs were carried out using a laboratory-developed real-time assay which used manual nucleic acid extraction ST-836 hydrochloride (Qiagen, Valencia, CA), primers and minor groove-binding hybridization probes from Epoch Biosciences (Bothell, WA), LightCycler Fast Start hybridization probe master mix (Roche, Indianapolis, IN), and the ABI HT7900 sequence detection system (Applied Biosystems, Foster City, CA). The assay targets a region of the P1 surface protein gene and has a limit of detection of 200 copies/ml. The assay targets a region of the major outer membrane protein gene and has a limit of detection of 320 copies/ml. The IgM ST-836 hydrochloride serologic testing for was performed by ELISA (values of 0.96 U/liter were interpreted as positive results), and for 0.001), yielding only 10 positive results out of 24,677 specimens (Table ?(Table1).1). Of 122 paired PCR and culture results, 3 were positive by PCR and none by culture. Of 285 patients for whom both IgM serology and culture performed, 19 were positive by serology and none by culture. Of the 280 prospectively collected, culture-negative specimens, none were positive when tested by PCR. TABLE 1. Total numbers of samples tested and percentages positive by the various methods used for and ST-836 hydrochloride diagnosis value 0.001), with no positive results in the 6,981 specimens submitted during the study period (Table ?(Table1).1). For 60 cases, both culture and IgM serology by ELISA were performed, 2 of which were positive by ELISA and none by culture. Of 154 cases for which both IgM serology by MIF and culture were performed, 4 were positive by serology and none by culture. There were an insufficient number of cases with both culture and PCR results for retrospective analysis. Of the 225 prospectively collected, culture-negative specimens, 2 were positive when tested by real-time PCR with crossing thresholds of 26.6 cycles (5.74 105 copies/ml) and 25.4 cycles (1.36 106 copies/ml), respectively. For diagnosis of acute and infections, few studies have focused on the utility of culture compared to other methods. The.