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Background Despite the World Health Organization (WHO)-reported elimination of measles in Australia, importation of cases especially in travellers from Asia continues in Sydney, Australia’s largest city. Laboratory confirmation supports clinico-epidemiological evidence of measles virus infection, and is needed to establish elimination. Objectives To evaluate the performance of a random access real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay using the moderate complexity SmartCycler ® platform, and measles antigen detection by immunofluorescence (IFA), for the detection of measles virus in patient samples. Study design One hundred samples comprising nose and throat swabs, nasopharyngeal aspirates and urine, collected from patients with suspected measles were tested in parallel using IFA and nucleic acid testing using the SmartCycler ® and LightCycler ® RT-PCR platforms. The LightCycler ® RT-PCR was used as the reference assay against which the SmartCycler ® RT-PCR and IFA were compared. Results Using the LightCycler ® RT-PCR, measles virus was detected in 35 clinical samples. There was 100% concordance between the results of the SmartCycler ® and the LightCycler ® -based RT-PCR. Measles genotypes detected included B3, D8, and D9. Testing urine in addition to NTS did not improve diagnostic yield. In contrast, the sensitivity and specificity of IFA compared to the reference LightCycler ® RT-PCR was 34.3% and 96.7%, respectively. Conclusion The performance of the SmartCycler ® is comparable to the LightCycler ® for the detection of measles virus. However, IFA had poor sensitivity and should not be used to confirm measles virus infection where nucleic acid testing is available. [ABSTRACT FROM AUTHOR]