Surgery and instruments: research

Why are these assays needed at all?

Validating an instrument reprocessing procedure against human prions requires a readout for human prions. Belondrade and colleagues state the constraint plainly: there is a lack of a rapid model permissive to human prions. Mouse bioassays with humanised lines run for hundreds of days per experiment, which is incompatible with product development cycles and with testing many conditions.

Their stated reason for addressing the problem is the lymphotropism of variant CJD prions combined with uncertainty about subclinical carriage. The 2016 paper cites the archived appendix survey estimate of roughly 1 in 2000 people in the United Kingdom positive for PrPTSE, and reasons from it that some surgical procedures carry a transmission risk in healthcare facilities.

What does Surf-PMCA measure?

The assay uses stainless steel wire as the carrier. Wires are contaminated with brain homogenate, dried, treated with the procedure under test, rinsed, then placed directly into a protein misfolding cyclic amplification reaction with normal brain homogenate from transgenic mice expressing ovine or human prion protein as substrate, run over several rounds of sonication and incubation with serial transfer, and read by proteinase K digestion and western blot. The quantity reported is residual seeding activity, expressed as the number of amplification rounds needed before signal appears.

Analytical performance in the 2016 report: specific detection of either human variant CJD or ovine scrapie PrPTSE adsorbed on a single steel wire at a 10 to the minus 8 brain dilution, within a two-week timeframe. Applied to reference and commercial prion-specific decontamination procedures, the efficiency of several marketed reagents at removing human variant CJD PrPTSE was lower than the authors expected.

Does residual seeding activity predict infectivity?

This is the question the 2020 mSphere paper was built to answer. Wires treated by different procedures were implanted intracerebrally in a transgenic mouse line permissive to variant CJD prions, and the in vivo outcome was compared with residual seeding activity measured in vitro. The authors report good concordance between the two, and state that the experiments emphasise the strength of Surf-PMCA as a rapid and sensitive assay for evaluating prion decontamination procedures, while confirming the lack of efficacy of several marketed reagents against variant CJD prions.

Good concordance is not identity. Individual wires were positive by Surf-PMCA with no corresponding disease in mice, which is the expected direction for a seeding assay that detects material below the infectious dose for that model. Two assumptions limit transfer to the clinic. The wires were air dried, while reusable devices in practice are often kept moist until reprocessing, and the tested commercial products had been approved using hamster 263K assays, a strain whose decontamination behaviour does not predict that of variant CJD prions.

What does the recombinant prion method add?

Erana and colleagues address a different weakness: natural brain isolates vary between donors, which makes repeat testing noisy. Their method uses recombinant prions produced in vitro, recovered from contaminated material by adherence to beads of different materials, with propagation read in vitro. They present it as technically simple, requiring a small shaker and standard biochemistry, reproducible between runs, and applicable to disinfectants of differing physicochemical nature.

The trade-off is in the test article. A recombinant prion of defined composition gives repeatability, while a clinical isolate carries the strain and the tissue matrix that an instrument would actually encounter. The two approaches answer different questions, and a procedure cleared by one has not been cleared by the other.

Which tissues should drive instrument policy?

Ocular surgery is the case where the assay evidence moved ahead of the guidance. Orru and colleagues measured post-mortem prion seeding activity by RT-QuIC in cornea, lens, ocular fluid, retina, choroid, sclera, optic nerve and extraocular muscle in a series of sporadic CJD eyes, covering three common subtypes. Seeding activity was detected in 100 percent of the eyes, with levels varying by up to 4 log-fold between individuals. The retina consistently carried the highest levels, in some cases only slightly below brain, and immunohistochemistry placed deposits in the retinal outer plexiform layer in most cases and the inner plexiform layer in some. Seed levels generally fell with distance from the brain, and yet every cornea had detectable seeds.

The authors note that sporadic CJD has been transmitted iatrogenically through corneal grafting, and that roughly 40 percent of patients develop visual or oculomotor symptoms and may present to an ophthalmologist first. Their conclusion covers both diagnostic potential and biohazard. Seeding activity is not a measurement of infectivity, and the samples are end-stage autopsy tissue, so the figures do not translate into a transmission probability for an instrument used on a living patient.

For the tissue distribution picture see tissue and organs, and for the plain-language versions surgery and instruments and its more detailed page. Related: food chain, chronic wasting disease and zoonotic risk and the regulations atlas.