Proper collection, handling, and transport of virologic specimens from suspected measles and rubella cases are crucial for successful virus detection and genetic characterization. The integrity of viral RNA may be compromised during any stage of specimen collection, processing, or shipment if temperature control is inadequate, repeated freeze–thaw cycles occur, or processing is delayed. To minimize RNA degradation, the procedures and recommendations outlined in Chapter 3 should be strictly followed.
The timing of specimen collection is equally critical for successful amplification and identification of measles or rubella RNA by any detection method. Specimens should be collected as soon as possible after rash onset, as the sensitivity of RNA detection decreases with time. The recommended timing for specimen collection is provided in Chapter 3. The sensitivity of RNA detection from most specimen types declines considerably after 5–7 days, although is generally detectable up to 15 days. However, different clinical specimens may still be collected beyond 5 days post-rash onset for detection of measles RNA if delays are unavoidable. Successful amplification of measles virus RNA by real-time RT-qPCR from oral fluid specimens collected up to 21 days after rash onset has been reported [2].
Oral fluid (OF), throat swabs (TS), or nasopharyngeal (NP) aspirates or swabs are good sources of RNA for measles or rubella virus detection [3,4,6]. TS and NP specimens are commonly used, particularly since the COVID-19 pandemic, and generally provide higher sensitivity for early RNA detection when collected within the first few days after rash onset. OF is also widely used, particularly in community settings, because of its non-invasive collection method, compatibility with both molecular and serologic testing, and its stability during transport and storage.
Urine samples are considered acceptable for measles virus confirmation but are less commonly used for rubella detection, except in cases of congenital rubella syndrome. They may be more difficult to transport due to their volume and refrigeration requirements and can contain substances that inhibit RT-qPCR if not processed properly. To improve detection sensitivity, the first-pass urine should be collected, centrifuged, and the pellet used for RNA extraction. When a suspected measles or rubella case is detected in an elimination setting, particularly when no source or epidemiological link is identified, collection of more than one clinical specimen may increase the likelihood of successful RNA detection and genotyping.
Serum specimens, including eluates from dried blood spots, have been used as a source of virus-specific RNA when standard virologic specimens could not be obtained. However, successful RNA detection in serum requires that blood is collected very close to rash onset, preferably within 3 days. Even when collected early, viremia is typically low, making RNA detection difficult.
Upon arrival at the laboratory, the package containing the clinical specimens for RT-qPCR testing should be inspected, and the condition of the specimens recorded (see Chapter 11, Data management and reporting of laboratory results). If not accessioned and processed immediately, specimens should be stored in a refrigerator (4° to 8 °C) or frozen at –70 °C, according to the standard operating procedures for the specimen type. Repeated freezing and thawing should be avoided to preserve RNA integrity.
The extraction of RNA from clinical specimens (or cell culture lysate) must be performed carefully to maximize yield and preserve integrity. Manual extraction using column-based kits (e.g. Qiagen QIAamp Viral RNA Mini or DSP) is widely used for serum, urine, swab, and CSF specimens. These protocols typically use 140–200 µL input and elute ~40–60 µL of RNA.
Many GMRLN laboratories also utilize automated magnetic bead–based systems for higher throughput and consistency. Common platforms include:
- Roche MagNA Pure instruments (LC 2.0, 96, Compact), processing up to 1 mL sample volume.
- bioMérieux NucliSENS easyMAG for efficient off-board lysis and extraction.
- Genes2Me Rapi X (Genes2Me Pvt. Ltd., Gurugram, India), used in some laboratories for rapid automated extraction following local validation.
Some laboratories are exploring advanced platforms such as the Panther Fusion (Hologic Inc., San Diego, CA, USA), on which laboratory-developed measles RT-qPCR assays have been validated with good performance [20], as well as emerging techniques such as RT-LAMP (Eiken Chemical Co., Ltd., Tokyo, Japan) [21] and microfluidic chip–based amplification for potential field use [22].
When selecting an extraction method, laboratory personnel must consider:
- The type and source of specimen (e.g. clinical versus cultured lysate)
- Required sample volume and throughput capacity
- Validation status of the method for measles or rubella detection
- Inclusion of internal and external controls to monitor extraction efficiency