Transmission of CJD from nasal brushings but not spinal fluid or RT-QuIC product
Gregory J. Raymond1 , Brent Race1 , Christina D. Orru 1 , Lynne D. Raymond1 , Matilde Bongianni2 , Michele Fiorini2 , Bradley R. Groveman1 , Sergio Ferrari2 , Luca Sacchetto3 , Andrew G. Hughson1 , Salvatore Monaco2 , Maurizio Pocchiari4 , Gianluigi Zanusso2 & Byron Caughey1 1 Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute for Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana 2 Department of Neurosciences, Biomedicine and Movement Sciences, University of Verona, Verona, Italy 3 Department of Surgical Sciences, Dentistry, Gynecology and Pediatrics, University of Verona, Verona, Italy 4 Department of Neuroscience, Istituto Superiore di Sanita, Rome, Italy
Abstract Objective: The detection of prion seeding activity in CSF and olfactory mucosal brushings using real-time quaking-induced conversion assays allows highly accurate clinical diagnosis of sporadic Creutzfeldt–Jakob disease. To gauge transmission risks associated with these biospecimens and their testing, we have bioassayed prion infectivity levels in patients’ brain tissue, nasal brushings, and CSF, and assessed the pathogenicity of amplified products of real-time quakinginduced conversion assays seeded with Creutzfeldt–Jakob disease prions. Methods: We obtained olfactory mucosal brushings and CSF from patients with a final diagnosis of sporadic Creutzfeldt–Jakob disease subtype MM1 (n = 3). Samples were inoculated intracerebrally into Tg66 transgenic mice that overexpress the homologous human 129M prion protein. The mice were evaluated for clinical, neuropathological, and biochemical evidence of prion infection. Results: Patients’ brain tissue at 102 to 105 fold dilutions affected 47/48 Tg66 mice. In contrast, maximum acutely tolerable doses of insoluble pellets from their olfactory mucosa brushings caused evidence of prion disease in only 4/28 inoculated mice, and no effects were seen with 10-fold dilutions. No clinical prion disease was observed in mice inoculated with antemortem CSF samples or prion-seeded real-time quaking-induced conversion assay products. Interpretation: Pellets from patients’ olfactory mucosa brushings had ≥10,000-fold lower infectivity per unit volume than brain tissue, while CSF lacked detectable infectivity. Nonetheless, the results suggest that appropriate precautions may be warranted in surgical interventions involving the olfactory areas. The lack of pathogenic infectivity in the real-time quaking-induced conversion assay products provides evidence that the assay does not replicate biohazardous prions in vitro.
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Discussion
Our current findings are relevant in the assessment of risks of tissue infectivity and sCJD transmission. As noted above, sCJD is well known to be transmissible following invasive iatrogenic exposures to prion infected tissues or neurosurgical procedures involving contaminated instruments.1 Divergent conclusions about risks of sCJD associated with extraneural surgical procedures have also been reported,21 and the strength of any such conclusions remain under debate. Importantly, other epidemiological studies have shown that health professionals in contact with CJD patients do not have enhanced occupational risk for CJD, suggesting that any occupational risks of sCJD infection via exposure in clinical settings may be negligible.22
With respect to CSF collected by lumbar puncture from sCJD patients, our humanized mouse bioassays did not detect levels of sCJD prions capable of causing disease despite the finding that these CSF samples were positive for prion seeding activity using the more sensitive RTQuIC assay. In contrast, as noted above, earlier transmission studies using nonhuman primates or other experimental animals have sometimes detected sCJD infectivity in CSF13-15. Brown et al. (13 and Dr. Paul Brown, personal communication) reported that CSF samples from 27 CJD cases were inoculated into 120 animals (65 nonhuman primates, 40 Guinea-pigs, and 15 other animal species) with successful transmissions for only four inocula (one Pan troglodytes, two Cebus capucinus, and one Cavia porcellus). It is likely that three of the four CSF samples that transmitted disease were collected postmortem (Dr. Paul Brown, personal communication). Postmortem degradation of brain tissues or exposure of the aspiration needle to brain tissue may have potentially contaminated CSF samples as has been suspected in comparisons of tau seeding activity in postmortem versus antemortem CSF specimens from tauopathy cases.23,24
Draganescu and coworkers also reported successful transmission to Guinea pigs (Cavia porcellus) for six of seven CSF samples from CJD patients,14 but the neuropathology of the diseased animals did not clearly show the characteristic spongiform changes of the cerebral grey matter, questioning the reliability of transmission. Finally, Tateishi and coworkers reported no transmission of an unspecified number of CSF samples from sCJD patients following intracerebral inoculation into mice.15 Another important uncertainty is the relative sensitivity of species of nonhuman primates and our Tg66 mice to sCJD. In comparing animal bioassay models, a feature that might be expected to enhance the susceptibility of Tg66 mice relative to primates and wild-type rodents is their overexpression of M129 human PrPC that is exactly sequencematched to our MM1 sCJD brain inocula. On the other hand, humans and other primates have much longer lifespans than rodents, giving more time for infections to progress to clinical disease. In any case, our side-by-side comparison of brain and CSF inocula in Tg66 mice indicated that CSF from living MM1 sCJD patients is at least several orders of magnitude less infectious than the same patients’ brain tissue. Moreover, our failure to cause disease by injecting relatively large volumes of sCJD CSF directly into the brain (typically the most efficient route) suggests that the transmission risks associated with incidental exposure to such CSF specimens may be negligible.
Consistent with our observations that sCJD OM samples often have 3–4 logs higher levels of prion seeding activity per µl than CSF samples,11 our bioassays indicated that our OM samples, and concentrated pools thereof, had sCJD infectivity that caused prion disease in a small minority of the recipient mice. Thus, OM infectivity was only slightly above the detection limit of the bioassay, which involved inoculation directly into the brain. Similarly, barely detectable levels of sCJD infectivity have been detected in plasma samples of two of four MM1 sCJD patients bioassayed in the humanized Tg340 mice mentioned earlier.17 Although our data indicate that there was higher infectivity in OM than CSF, the OM infectivity levels were again orders of magnitude lower than in brain tissue. Nonetheless, immunochemical analyses of the olfactory neuroepithelium of human CJD cases showed that the deposits of the pathological prion protein were unevenly distributed in the cilia.25 Taken together, these findings suggest that the olfactory area of the nasal vault represents a site of potential risk, albeit relatively low, in mediating iatrogenic CJD transmission. Therefore, instruments used for nasal vault surgery should be considered for prion decontamination procedures or disposal. One study reported that people having experienced nose or throat surgery, without specifying the specific surgery of each subject, had three times the risk of sCJD compared to the general population.26 Additional and more detailed studies are required to better estimate the risk of surgical procedures involving the nasal vault. A limitation of this study is that other individual MM1 sCJD cases, and cases with other sCJD strains27 besides the most common MM1 sCJD subtype that we have evaluated here, might give different levels of infectivity in OM brushings or mucus. Further transmission studies should provide broader perspective on this issue.
The fact that pathogenic sCJD prions do not amplify in our RT-QuIC reactions using recombinant hamster PrPC 90-231 should help to allay fears of additional biohazard being generated in conducting these tests. However, we note that some RT-QuIC assays for human prions use full-length hamster, human, bank vole, or other recombinant PrPC substrate,28 and one cannot conclude from our current data that RT-QuIC products generated with other PrPC substrates or under other assay conditions are as safe as those generated with hamster PrPC 90-231.
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