Laboratory methods supporting measles surveillance in Queensland, Australia, 2010–2017

Abstract

Purpose. Australia was officially recognised as having eliminated endemic measles transmission in 2014. Maintaining laboratory support for surveillance of vaccine-preventable diseases, such as measles, is an essential component of reaching and maintaining transmission-free status. Methodology. Real-time and conventional PCR-based tools were used to detect, differentiate from measles vaccine virus (MeVV), and sequence fragments of measles viruses (MeV) identified from specimens collected in Queensland. Specimens were mostly from travellers who had visited or returned to Queensland from international or interstate sites or been in contact with a case from either group. Results. Between 2010 and 2017, 13 678 specimens were tested in our laboratory using real-time RT-PCR (RT-rPCR), identifying 533 positives. Most specimens were swabs (70.98 %) and urines (25.56 %). A MeVV RT-rPCR was used on request and identified 154 instances of MeVV. MeV-positive extracts were genotyped as required. Genotypes identified among sequenced specimens included B3, D4, D8, D9, G3, and H1 as well as members of clade A as expected from the detection of MeV among virus introductions due to global travel and vaccination. Conclusion. We describe the workflow employed and results from our laboratory between 2010 and 2017 for the sensitive detection of MeV infection, supporting high-quality surveillance to ensure the maintenance of Australia’s measles-free status.

Molecular Detection of Measles Virus from Febrile Rash Illness Cases in Lagos State, Nigeria

“…Samples were processed immediately after collection, but in case they were not processed immediately, they were stored at -20°C. Each stored sample was tested for MV [19] within one week from time of collection.…”

“…Ribonucleic Acid (RNA) extraction was performed using method previously described by McMahon et al [19], with the process conducted inside a biosafety cabinet class II.…”

Measles vaccine virus RNA in children more than 100 days after vaccination

“…Upon receipt of specimens with a request for MeV PCR, total RNA was extracted using the Qiagen EZ1 Mini kit v 2.0 (Qiagen, Hilden, Germany). Five of the 90µl elution was added to a measles virus (MeV) real-time reverse transcription-polymerase chain reaction (RT-rPCR) targeting the fusion (F) gene and designed to detect all MeV genotypes [9]. Nucleic acids were re-extracted from all MeV positive samples and testing was repeated using the MeV RT-rPCR.…”

“…The primer, probes, and cycling conditions for the MeV and the MeVV RT-rPCR assays have been previously described [9]. The MeV RT-rPCR was used with a final primer concentration of 0.3μM and probe concentration of 0.15μM.…”

“…Synthetic probe and primer controls were used for both RT-rPCR assays, alongside no template controls (NTC) and negative extraction controls [9]. A threshold cycle (CT) value of Raw sequence data were cleaned, forward and reverse strands were aligned, and primer sequences removed.…”