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Gut. 2007 September; 56(9): 1329–1331.
PMCID: PMC1954950

A non‐invasive screen for infectivity in transmissible spongiform encephalopathies

The risk of transmissible spongiform encephalopathy (TSE) infection from endoscopic procedures has been discussed recently in this journal.1,2 Subclinical carriers of variant Creutzfeldt–Jakob disease (vCJD), the numbers of whom are unknown, may present a risk of iatrogenic infection to susceptible patients participating in endoscopy. We report a pilot study, undertaken here on sheep, describing a non‐invasive assessment of autonomic function based on heart rate variability (HRV) which may provide a useful screening method for subclinical carriers of certain TSE diseases.

Detection of disease associated prion protein (PrPd) in the brain stem is used in confirmatory postmortem tests for bovine spongiform encephalopathy (BSE) and sheep scrapie; this protein is also present in humans with vCJD.3,4,5 In scrapie, the timing of PrPd accumulation in the dorsal motor nucleus of the vagus nerve (DMNX) indicates that this is a likely site of initial neuroinvasion.4 Non‐myelinated vagal fibres from the DMNX and faster myelinated vagal fibres from the nucleus ambiguus are involved in the control of HRV, which is the millisecond beat to beat change that is distinct from the heart rate. Cardiac vagal tone may be considered to be the sum of low frequency (LF) variation in HRV influenced by vagal efferents from the DMNX, plus high frequency (HF) variations, influenced by the respiratory frequency, resulting from the efferent output from the nucleus ambiguus.6 The DMNX contains parasympathetic neurones whose axons communicate directly with the gut, heart, and other viscera and can be implicated in the transport and initial site of infection and subsequent infection in the nucleus tractus solitarius and the nucleus ambiguus.4 Thus analysis of HRV may provide an index of vagal dysfunction caused by the presence of PrPd in brain stem regions before clinical signs become noticeable.

PrPd has been detected in vCJD rectal samples and implications for potential iatrogenic infection to humans of susceptible genotypes have been drawn.1,2 Large scale studies of sheep scrapie show that PrPd aggregates are consistently present in the recto‐anal‐mucosa‐associated lymphoid tissue (RAMALT) of preclinically and clinically infected sheep.7,8 This pilot study investigated the possibility that HRV might also recognise TSE infection in clinically normal sheep, positive for PrPd in RAMALT biopsies.biopsies.

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Table 1 Summary of the genotypes of the sheep involved in the study

We recorded HRV data from 25 sheep. Six were clinically normal but PrPd positive in RAMALT biopsies, and 19 were clinically normal and PrPd negative in RAMALT biopsies. Among the latter, three were unchallenged controls, 11 were naturally exposed to scrapie infection and of resistant PrP genotypes, one was naturally exposed to scrapie and of susceptible genotype, and four were orally dosed and of susceptible genotypes. Experimentally infected sheep had been dosed with 5 g of a 10% homogenate of a pool from scrapie affected sheep brains.

ECG samples (300 s) were collected and digitised using a commercially available monitor (VariaCardio). ECG R wave timings were obtained to determine variability in the R–R intervals, and an instantaneous tachygram was constructed (fig 11)) from which power spectra were calculated.

figure gt123141.f1
Figure 1 Typical high frequency (HF) and low frequency (LF) filtered tachygrams on which a fast Fourier transformation is done to give estimates of heart rate variability (HRV) in different frequency bands. The reduced HRV is indicated by the ...

Power spectral analysis in the 0.032 to 0.138 Hz band (LF), but not the 0.15 to 0.5 Hz band (HF) showed significant differences (LF, p<0.005) between RAMALT biopsy negative and biopsy positive sheep (fig 22).). As all positive sheep were asymptomatic, HRV assessment may be a useful preclinical test for TSE infection. This HRV index on its own, used once, may not distinguish between individual rectal biopsy positive animals and controls, as ranges overlapped. However, the negative and positive biopsy groups contained animals of different provenance—the negative biopsy group contained sheep of resistant genotypes, unexposed sheep, and sheep of uncertain infection status; the positive group contained infected sheep at approximately 40–90% of the incubation period. Owing to the small number of sheep investigated here, further large scale studies are needed to refine the methods and aid the interpretation of the results; however, being a live non‐invasive screen for TSE infection it has the advantage that repeated measures over time may be taken to strengthen confidence in the interpretation.

figure gt123141.f2
Figure 2 Box plots (median, upper and lower quartiles, and range) showing heart rate variability (HRV) measures, demonstrating a significant difference (**p<0.005) between biopsy positive and biopsy negative animals (Mann–Whitney ...

We have previously demonstrated a similar reduction in HRV in BSE infected cattle9 and in humans incubating vCJD10 compared with controls. We herewith show that changes in HRV may be a common and early feature of TSE infections. Improved preclinical testing for TSEs using specific signature changes in HRV with respect to time could help minimise the risk of iatrogenic infection from endoscopy and other invasive procedures, and also provide an objective measure of the pathogenesis of the disease.

Footnotes

Conflict of interest: None declared.

References

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