Wide distribution of prion infectivity in the peripheral tissues of vCJD and sCJD patients
Published: 02 February 2021
Volume 141, pages 383–397, (2021)
Jean-Yves Douet, Alvina Huor, Hervé Cassard, Séverine Lugan, Naima Aron, Mark Arnold, Didier Vilette, Juan-Maria Torres, James W. Ironside & Olivier Andreoletti
Abstract
Sporadic Creutzfeldt-Jakob disease (sCJD) is the commonest human prion disease, occurring most likely as the consequence of spontaneous formation of abnormal prion protein in the central nervous system (CNS). Variant Creutzfeldt–Jakob disease (vCJD) is an acquired prion disease that was first identified in 1996. In marked contrast to vCJD, previous investigations in sCJD revealed either inconsistent levels or an absence of PrPSc in peripheral tissues. These findings contributed to the consensus that risks of transmitting sCJD as a consequence of non-CNS invasive clinical procedures were low. In this study, we systematically measured prion infectivity levels in CNS and peripheral tissues collected from vCJD and sCJD patients. Unexpectedly, prion infectivity was detected in a wide variety of peripheral tissues in sCJD cases. Although the sCJD infectivity levels varied unpredictably in the tissues sampled and between patients, these findings could impact on our perception of the possible transmission risks associated with sCJD.
snip…
Recently, bioassays in transgenic mice that express the human PrP gene and display a high sensitivity to sCJD demonstrated the presence of prion infectivity in the plasma and bone marrow of several sCJD patients [17, 32]. These results raised questions about the overall distribution of prion infectivity and transmission risks associated with other peripheral tissues from sCJD patients.
In this study, we measured the prion infectivity levels in a panel of tissues collected from vCJD and sCJD MM1 cases. These bioassays demonstrated that, as expected, consistent titres of infectivity were present in lymphoid tissues from vCJD patients. However, for the non-lymphoid peripheral tissues studied, variable and lower titres of infectivity were detected in both sCJD and vCJD patients. These findings could impact on our perception of the possible transmission risks associated with sCJD involving non-CNS invasive procedures.
snip…
Unexpectedly, in the majority of the cases, the inoculation in tgMet of the peripheral tissues from the same MM1 sCJD patients resulted in positive transmission. TgMet inoculation revealed the presence of prion infectivity in all the different categories of peripheral tissues except liver and gonads (Table 3).
However, in contrast with vCJD, the bioassay results (positive versus absence of transmission) indicated that the prion infectivity distribution pattern in peripheral tissues strongly differed between the five affected MM1 sCJD patients. For instance, in sCJD case 2, positive transmissions were observed in tgMet inoculated with 10 out of the 13 tested peripheral tissues including lymphoid tissues, salivary glands, kidney, heart and pancreas. In contrast, in sCJD case 3, positive transmissions were only observed for 4 out of the same 13 tissues (lung, adrenal gland, bone marrow and skeletal muscle), with no disease transmission resulting from inoculation of lymphoid tissues, salivary gland, kidney, heart or pancreas (Table 3).
Interestingly, bioassay of the lymphoid tissues (spleen, cervical lymph node and tonsil) resulted in a disease transmission in only 3 out of the 5 sCJD cases (cases 2, 4 and 5). These results support the contention that in MM1 sCJD patients, the presence / absence of infectivity in the lymphoid organs is apparently not a determinant driver of the accumulation of infectivity in the other categories of peripheral tissues.
Despite the differences in bioassay transmission patterns, the PrPres Western blot profile and the vacuolar lesions profiles overserved in tgMet mice inoculated with MM1 sCJD peripheral tissues and frontal cortex brain homogenates were identical (Fig. 2), indicating that the same prion strain was present in the peripheral tissues and the CNS of the five MM1 sCJD patients.
No transmission or PrPres accumulation was observed in tgMet mice inoculated with peripheral tissues from the non CJD control patient (Table 3, Fig. 2).
Infectivity titres estimates In order to estimate the infectivity levels in the vCJD and MM1 sCJD patients’ tissues, we applied the method described by Arnold et al. [4]. This approach uses both the probability of survival (attack rate at each dilution) and the individual mouse survival time at each dilution. The relationship between the titre of inoculum and the probability of infection and the length of the survival times were derived from data corresponding to endpoint titration of a vCJD and a MM1 reference isolate in tgBov and tgMet mice, respectively [17, 32]. A normal distribution for the relationship between dose and survival time was assumed and the probability of infection versus dose was assumed to follow a logistic regression curve (supplementary Fig. 1).
Using this approach, the infectious titre in the frontal cortex of the four vCJD patients was estimated to range between 106.11 and 106.74 ID50 IC in tgBov per gram of tissue (Table 2).
The estimated infectivity levels in secondary lymphoid tissues (spleen and cervical lymph node) were 1 to 4 log10 lower than in the frontal cortex of the same patient. Infectivity levels in the other categories of peripheral tissues were 2.5 to 6 log10 lower than in the frontal cortex (Table 2, Fig. 3).
Strikingly, in some of the vCJD affected patients, infectivity levels in heart (vCJD-3 and 4), kidney (vCJD-3 and 4), lung(vCJD-3), salivary gland (vCJD-3 and 4) or thyroid (vCJD-4) were only 1 to 2 log10 lower than in the spleen and/or cervical lymph node (Table 2, Fig. 3).
As already stated, the distribution of the prion in the peripheral tissue of the vCJD asymptomatic case was more restricted than in vCJD affected patients (Fig. 3). However, when positive, the peripheral tissues from the asymptomatic patient displayed similar infectivity levels to those observed in the vCJD patients at the clinical stage of the disease (Table 2, Fig. 3).
In the MM1 sCJD patients, the estimated infectivity levels in the frontal cortex varied between 105.8 and 107.8 ID50 IC in tgMet/gram (Table 3). Infectivity levels in peripheral tissues that scored positive in bioassay were 2.8 to 8 log10 lower than in the frontal cortex of the same patient (Table 3, Fig. 3). In all five cases, no obvious relationship seemed to exist between the infectivity level in the CNS (frontal cortex) and either the distribution or the levels of sCJD infectivity in their peripheral tissues (Fig. 3).
In the sCJD cases 2, 4 and 5, the lymphoid tissues displayed maximal level of infectivity that were 3–3.5 log10 lower than those observed in the frontal cortex (Table 2, Fig. 3). Strikingly, the infectivity levels associated with some peripheral tissues such as salivary gland (sCJD2), heart (sCJD2), kidney (sCJD 2) or bone marrow (sCJD 5) could be equivalent or even higher than those measured in the lymphoid organs of the same patient (Table 2, Fig. 3).
Discussion
vCJD associated risks Following the emergence of vCJD in the UK in 1996, the presence of abnormal prion protein in the lymphoid tissues of affected patients was rapidly identified [31, 55]. This immediately raised major concerns about the risk of its iatrogenic transmission of the disease (via contaminated surgical instruments and blood transfusion) and led, in many countries, to the implementation of specific preventing against this risk.
Despite a relatively limited number of identified clinical cases (n = 231), the most recent epidemiological studies indicated that 1 out 2000 people in the UK could carry the vCJD agent (as judged by the presence of abnormal prion protein detected by immunohistochemistry in lymphoid follicles in the appendix) and that the exposure period to BSE agent in the UK could have largely exceeded the period initially considered to be at risk (i.e. the 1985–1996 period) [22, 23]. Over the 25 years since the emergence of vCJD only five instances that are a likely consequence of iatrogenic vCJD transmission have come to light, all in the UK and all associated with blood and blood-products [36, 37, 46, 47]. No cases of vCJD due to iatrogenic transmission by medical or surgical procedures have been identified, indicating that the preventive measures implemented to mitigate its transmission were effective [26, 39].
The overall picture of the distribution and levels of prion infectivity in the tissues of both vCJD affected and asymptomatic patients that we here report confirm the numerous hypotheses and the mosaic of experimental data that were used to design the infection control measures that were successful in limiting iatrogenic vCJD transmissions. These results also reinforce the fact that vCJD transmission risks have not disappeared since each asymptomatic vCJD-infected individual in a human population will continue to represent a potential source of disease transmission.
Peripheral tissue infectivity in sCJD Seminal transmission experiments of peripheral tissues from sCJD cases in primate models failed to detect infectivity in a large selection of peripheral tissues, body fluids and excretions (except in one liver sample) [10]. Abnormal PrP immunodetection techniques (Western blot and immunohistochemistry) also failed to reveal the presence of prion in the peripheral tissues of sCJD patients [27, 28].These findings led to the generally accepted view that prion infectivity in the sCJD remains mostly confined to the CNS.
In 2003, improved Western Blot protocols for PrPres immunodetection revealed the presence of prion in the spleen (10 positive out of 28 cases) and/or the skeletal muscle (8 positive out of 32 cases) [24]. More recently, transmission studies of sCJD tissues in human PrP expressing transgenic mice (using plasma and bone marrow samples) and in vitro amplification of prions in a small number of sCJD peripheral tissues (skin, kidney, lung, adrenal gland) provided further evidence to the view that prions can accumulate in the peripheral tissues of sCJD affected patients [17, 32, 43, 52].
The results that we here report provide unequivocal and definitive evidence of the widespread distribution of the prion infectivity in the peripheral tissues in MM1 sCJD patients.
Other types of patients Since our study was restricted to MM1 sCJD cases (the commonest sCJD subtype), additional investigations will be necessary to formally establish that consistent accumulation of prions in peripheral tissues also occurs in patients with other PRNP genotypes (Met/Val129 and Val/Val129) and/or affected with other sCJD prion strains [11]. The presence of abnormal PrP (in spleen and the skeletal muscle) and infectivity (in bone marrow) already identified in MV2 and VV2 sCJD patients indicates that this phenomenon is unlikely to be limited to MM1 sCJD cases [24, 32].
Variability in sCJD patients The nature of peripheral tissues that accumulated infectivity in sCJD and vCJD cases were relatively similar. While the patterns of prion distribution and the infectivity levels observed in the peripheral tissues were relatively homogenous in the vCJD cases, particularly in lymphoid tissues, a high degree of variability was observed across the sCJD cases. Brain vacuolar lesion profiling and PrPres WB typing confirmed that the same prion strain was present in the brain and the peripheral tissues of all the sCJD affected patients. This rules out the hypothesis that prion strain(s) difference(s) (between individuals or in tissues from a same individual) could be responsible for the observed variability.
The comparison of prion distribution pattern in sCJD case 2 (20-months clinical phase duration) and sCJD cases 1, 3, 4 and 5 patients (1–4 months clinical phase duration) might suggest that, at first glance, a longer clinical phase duration is likely to be associated with a more widespread distribution of prions in the body tissues. However, even if our study represents an unprecedented effort for characterizing CJD agent distribution patterns in the organs of affected patients, the number of sCJD cases that we investigated is too limited to draw definitive conclusions. The characterisation of a larger cohort of sCJD patients will be necessary to establish the relationship that might exist between the distribution and/or infectivity levels in peripheral tissues and sCJD patient age at clinical onset and the duration of the clinical phase of the illness.
Iatrogenic transmission of CJD Several hundred cases of iatrogenic CJD transmission (iCJD) have been reported worldwide, the vast majority of which are likely to represent transmissions from sCJD patients [9]. The principal sources of these outbreaks were intramuscular injections with contaminated human pituitary-derived growth hormone (226 cases) and implantation of dura mater grafts (228 cases) derived from human cadavers with undiagnosed sCJD infections. A small number of cases were apparently also caused by neurosurgery using contaminated neurosurgical instruments and EEG electrodes (6 cases), transplantation of corneal grafts (2 cases) and intramuscular injections with human pituitary-derived gonadotrophic hormone (4 cases) [9].
These cases dramatically illustrate the high resistance of CJD prions to standard medical decontamination procedures and their particular abilities to bind to steel surgical instruments [19, 53]. Survival times in the individuals who were exposed to sCJD agent(s) by the peripheral route could be extremely long and variable; for instance, in patients that received intramuscular injection of contaminated human pituitary-derived growth hormone, the onset of clinical signs and symptoms of iatrogenic CJD could be observed between 4 and 42 years after the treatment [9].
In a context where sCJD infectivity is apparently limited to the CNS, the medical and surgical procedures responsible for iatrogenic transmission of the disease remain relatively limited, the overall risks for sCJD iatrogenic transmission are now considered to be remote since most of these established routes of transmission be avoided (e.g. the use of human pituitary-derived hormones and human dura mater grafts) [9].
Our detection of low levels of sCJD infectivity in non-CNS tissues such as lung, heart, muscle or even salivary gland was unexpected. We next reviewed the medical histories for the sCJD patients that we studied (Table 1), which revealed surgical procedures (s-CJD-2 and 4) and/or invasive medical examinations (polypectomy under colonoscopy sCJD-5) only few years before the clinical disease in these patients. Some years ago, the presence of infectivity in the plasma and the detection of abnormal prion protein in the urine of sCJD patients raised concerns about the risks of sCJD transmission by blood transfusion and plasma/urine-derived medical products [17, 38]. The detection, in our study, of infectivity in the bone marrow and the kidney of sCJD patients further reinforces these concerns.
Prion titres as measured by intracerebral inoculation in PrP over-expressing transgenic mice models can provide us with estimates of the relative infectivity levels present in the CNS and peripheral tissues of affected patients. This approach conforms to the current gold standard for quantification of prions. However, in a context where the amount of infectivity that would be necessary to transmit disease to another human remains unknown, the infectivity titre estimates (as established following intracerebral inoculation in a reporter animal model) cannot be used to directly infer transmission risks to patients. Other factors, including the various different potential exposure routes (subcutaneous, intramuscular, etc.) and the decontamination/sterilisation methodologies used on the surgical instruments /materials involved, will also influence the transmission risk analysis [1].
In many industrialized countries, reliable CJD passive surveillance programs have been established for decades. The apparently stable and low prevalence of sCJD cases in these countries bring some reassurance about the low numbers of iatrogenic CJD cases in this century [9], perhaps reflecting the variable and lower prion titres detected in the non-CNS tissues in this study.
Guidelines are in place to mitigate and control the risk of iatrogenic transmission of CJD in a healthcare setting [1, 56]. The procedures for cleaning and decontamination of surgical instruments and/or medical equipment now includes an assessment of their potential contamination by prions. However, prions are notoriously resistant to physico/chemical treatments and decontamination process that would be efficient on these agents remain generally inapplicable to some surgical and medical equipment [54], resulting in recommendations to destroy neurosurgical instruments that have been used on the brain of a patient with definite or probable CJD. Furthermore, the presence of dementia or an evolving neurodegenerative disorder in a patient undergoing medical or surgical procedures triggers the use of specific protocols designed to prevent the risk of potential transmission of CJD from the equipment used (surgical tools, endoscopes, etc.). The use of cells/tissues/organs and body fluids from these groups of patients for therapeutic purposes (blood donations, tissue grafts, etc.) is also restricted [1, 56].
When considering the risk of iatrogenic transmission of prion agents from CJD patients, one fundamental question is: How early before the occurrence of clinical signs and symptoms is prion infectivity likely to be present in peripheral solid tissues, blood and urine?
All the samples that we used to establish infectivity levels in tissues were collected post mortem in sCJD and vCJD patients at the terminal stage of the disease. There is clear evidence for the presence of infectivity in the blood and the peripheral tissues years before the clinical onset of vCJD in asymptomatic infected patients [3, 8, 16, 17]. However, it is uncertain that the infectivity levels and distribution in the post mortem peripheral tissues of sCJD and vCJD patients reflect the situation that could be observed at a preclinical or early clinical stage in the same patients. In the absence of tissue samples collected from asymptomatic sCJD patients we are totally lacking in data, therefore trying to elaborate further on this question would be totally speculative.
In several forms of genetic prion disease, for example those associated with the E200K PRNP mutation, the clinical disease manifestations, PrPres WB signature and tissue PrPres distributions are similar to that in sCJD [34, 48]. Despite the ethical issues it might raise, the longitudinal collection of blood samples and body fluids, for research purposes, in consenting patients belonging to families affected by these genetic forms of prion diseases (with confirmed mutations of the PRNP gene) may represent the only possibility to address this question.
In conclusion, the systematic surveillance of CJD and related epidemiological studies in many countries confirm the decline of cases of iatrogenic CJD due to recognised medical or surgical procedures, such as human dura mater graft surgery or treatment with human pituitary-derived growth hormone. However, they do not exclude the possibility that iatrogenic transmission could at least partly account for some sCJD cases observed in the population, particularly in localized geographic regions with evidence of CJD case clusters [50].
Many uncertainties remain on the early stages of the prion accumulation and infectivity in the peripheral tissues in patients infected with sCJD. However, the results of this study suggest that the iatrogenic transmission risks associated with sCJD peripheral tissues should not be disregarded.
Data and materials availability
All data are presented in the manuscript and the supplementary information. Human biological material used in the study was provided by the National CJD Research & Surveillance Unit Brain and Tissue Bank in Edinburgh, UK, which is part of the Edinburgh Brain and Tissue Bank supported by the Medical Research Council.
References
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Published: 02 February 2021
Volume 141, pages 383–397, (2021)
Jean-Yves Douet, Alvina Huor, Hervé Cassard, Séverine Lugan, Naima Aron, Mark Arnold, Didier Vilette, Juan-Maria Torres, James W. Ironside & Olivier Andreoletti
Abstract
Sporadic Creutzfeldt-Jakob disease (sCJD) is the commonest human prion disease, occurring most likely as the consequence of spontaneous formation of abnormal prion protein in the central nervous system (CNS). Variant Creutzfeldt–Jakob disease (vCJD) is an acquired prion disease that was first identified in 1996. In marked contrast to vCJD, previous investigations in sCJD revealed either inconsistent levels or an absence of PrPSc in peripheral tissues. These findings contributed to the consensus that risks of transmitting sCJD as a consequence of non-CNS invasive clinical procedures were low. In this study, we systematically measured prion infectivity levels in CNS and peripheral tissues collected from vCJD and sCJD patients. Unexpectedly, prion infectivity was detected in a wide variety of peripheral tissues in sCJD cases. Although the sCJD infectivity levels varied unpredictably in the tissues sampled and between patients, these findings could impact on our perception of the possible transmission risks associated with sCJD.
snip…
Recently, bioassays in transgenic mice that express the human PrP gene and display a high sensitivity to sCJD demonstrated the presence of prion infectivity in the plasma and bone marrow of several sCJD patients [17, 32]. These results raised questions about the overall distribution of prion infectivity and transmission risks associated with other peripheral tissues from sCJD patients.
In this study, we measured the prion infectivity levels in a panel of tissues collected from vCJD and sCJD MM1 cases. These bioassays demonstrated that, as expected, consistent titres of infectivity were present in lymphoid tissues from vCJD patients. However, for the non-lymphoid peripheral tissues studied, variable and lower titres of infectivity were detected in both sCJD and vCJD patients. These findings could impact on our perception of the possible transmission risks associated with sCJD involving non-CNS invasive procedures.
snip…
Unexpectedly, in the majority of the cases, the inoculation in tgMet of the peripheral tissues from the same MM1 sCJD patients resulted in positive transmission. TgMet inoculation revealed the presence of prion infectivity in all the different categories of peripheral tissues except liver and gonads (Table 3).
However, in contrast with vCJD, the bioassay results (positive versus absence of transmission) indicated that the prion infectivity distribution pattern in peripheral tissues strongly differed between the five affected MM1 sCJD patients. For instance, in sCJD case 2, positive transmissions were observed in tgMet inoculated with 10 out of the 13 tested peripheral tissues including lymphoid tissues, salivary glands, kidney, heart and pancreas. In contrast, in sCJD case 3, positive transmissions were only observed for 4 out of the same 13 tissues (lung, adrenal gland, bone marrow and skeletal muscle), with no disease transmission resulting from inoculation of lymphoid tissues, salivary gland, kidney, heart or pancreas (Table 3).
Interestingly, bioassay of the lymphoid tissues (spleen, cervical lymph node and tonsil) resulted in a disease transmission in only 3 out of the 5 sCJD cases (cases 2, 4 and 5). These results support the contention that in MM1 sCJD patients, the presence / absence of infectivity in the lymphoid organs is apparently not a determinant driver of the accumulation of infectivity in the other categories of peripheral tissues.
Despite the differences in bioassay transmission patterns, the PrPres Western blot profile and the vacuolar lesions profiles overserved in tgMet mice inoculated with MM1 sCJD peripheral tissues and frontal cortex brain homogenates were identical (Fig. 2), indicating that the same prion strain was present in the peripheral tissues and the CNS of the five MM1 sCJD patients.
No transmission or PrPres accumulation was observed in tgMet mice inoculated with peripheral tissues from the non CJD control patient (Table 3, Fig. 2).
Infectivity titres estimates In order to estimate the infectivity levels in the vCJD and MM1 sCJD patients’ tissues, we applied the method described by Arnold et al. [4]. This approach uses both the probability of survival (attack rate at each dilution) and the individual mouse survival time at each dilution. The relationship between the titre of inoculum and the probability of infection and the length of the survival times were derived from data corresponding to endpoint titration of a vCJD and a MM1 reference isolate in tgBov and tgMet mice, respectively [17, 32]. A normal distribution for the relationship between dose and survival time was assumed and the probability of infection versus dose was assumed to follow a logistic regression curve (supplementary Fig. 1).
Using this approach, the infectious titre in the frontal cortex of the four vCJD patients was estimated to range between 106.11 and 106.74 ID50 IC in tgBov per gram of tissue (Table 2).
The estimated infectivity levels in secondary lymphoid tissues (spleen and cervical lymph node) were 1 to 4 log10 lower than in the frontal cortex of the same patient. Infectivity levels in the other categories of peripheral tissues were 2.5 to 6 log10 lower than in the frontal cortex (Table 2, Fig. 3).
Strikingly, in some of the vCJD affected patients, infectivity levels in heart (vCJD-3 and 4), kidney (vCJD-3 and 4), lung(vCJD-3), salivary gland (vCJD-3 and 4) or thyroid (vCJD-4) were only 1 to 2 log10 lower than in the spleen and/or cervical lymph node (Table 2, Fig. 3).
As already stated, the distribution of the prion in the peripheral tissue of the vCJD asymptomatic case was more restricted than in vCJD affected patients (Fig. 3). However, when positive, the peripheral tissues from the asymptomatic patient displayed similar infectivity levels to those observed in the vCJD patients at the clinical stage of the disease (Table 2, Fig. 3).
In the MM1 sCJD patients, the estimated infectivity levels in the frontal cortex varied between 105.8 and 107.8 ID50 IC in tgMet/gram (Table 3). Infectivity levels in peripheral tissues that scored positive in bioassay were 2.8 to 8 log10 lower than in the frontal cortex of the same patient (Table 3, Fig. 3). In all five cases, no obvious relationship seemed to exist between the infectivity level in the CNS (frontal cortex) and either the distribution or the levels of sCJD infectivity in their peripheral tissues (Fig. 3).
In the sCJD cases 2, 4 and 5, the lymphoid tissues displayed maximal level of infectivity that were 3–3.5 log10 lower than those observed in the frontal cortex (Table 2, Fig. 3). Strikingly, the infectivity levels associated with some peripheral tissues such as salivary gland (sCJD2), heart (sCJD2), kidney (sCJD 2) or bone marrow (sCJD 5) could be equivalent or even higher than those measured in the lymphoid organs of the same patient (Table 2, Fig. 3).
Discussion
vCJD associated risks Following the emergence of vCJD in the UK in 1996, the presence of abnormal prion protein in the lymphoid tissues of affected patients was rapidly identified [31, 55]. This immediately raised major concerns about the risk of its iatrogenic transmission of the disease (via contaminated surgical instruments and blood transfusion) and led, in many countries, to the implementation of specific preventing against this risk.
Despite a relatively limited number of identified clinical cases (n = 231), the most recent epidemiological studies indicated that 1 out 2000 people in the UK could carry the vCJD agent (as judged by the presence of abnormal prion protein detected by immunohistochemistry in lymphoid follicles in the appendix) and that the exposure period to BSE agent in the UK could have largely exceeded the period initially considered to be at risk (i.e. the 1985–1996 period) [22, 23]. Over the 25 years since the emergence of vCJD only five instances that are a likely consequence of iatrogenic vCJD transmission have come to light, all in the UK and all associated with blood and blood-products [36, 37, 46, 47]. No cases of vCJD due to iatrogenic transmission by medical or surgical procedures have been identified, indicating that the preventive measures implemented to mitigate its transmission were effective [26, 39].
The overall picture of the distribution and levels of prion infectivity in the tissues of both vCJD affected and asymptomatic patients that we here report confirm the numerous hypotheses and the mosaic of experimental data that were used to design the infection control measures that were successful in limiting iatrogenic vCJD transmissions. These results also reinforce the fact that vCJD transmission risks have not disappeared since each asymptomatic vCJD-infected individual in a human population will continue to represent a potential source of disease transmission.
Peripheral tissue infectivity in sCJD Seminal transmission experiments of peripheral tissues from sCJD cases in primate models failed to detect infectivity in a large selection of peripheral tissues, body fluids and excretions (except in one liver sample) [10]. Abnormal PrP immunodetection techniques (Western blot and immunohistochemistry) also failed to reveal the presence of prion in the peripheral tissues of sCJD patients [27, 28].These findings led to the generally accepted view that prion infectivity in the sCJD remains mostly confined to the CNS.
In 2003, improved Western Blot protocols for PrPres immunodetection revealed the presence of prion in the spleen (10 positive out of 28 cases) and/or the skeletal muscle (8 positive out of 32 cases) [24]. More recently, transmission studies of sCJD tissues in human PrP expressing transgenic mice (using plasma and bone marrow samples) and in vitro amplification of prions in a small number of sCJD peripheral tissues (skin, kidney, lung, adrenal gland) provided further evidence to the view that prions can accumulate in the peripheral tissues of sCJD affected patients [17, 32, 43, 52].
The results that we here report provide unequivocal and definitive evidence of the widespread distribution of the prion infectivity in the peripheral tissues in MM1 sCJD patients.
Other types of patients Since our study was restricted to MM1 sCJD cases (the commonest sCJD subtype), additional investigations will be necessary to formally establish that consistent accumulation of prions in peripheral tissues also occurs in patients with other PRNP genotypes (Met/Val129 and Val/Val129) and/or affected with other sCJD prion strains [11]. The presence of abnormal PrP (in spleen and the skeletal muscle) and infectivity (in bone marrow) already identified in MV2 and VV2 sCJD patients indicates that this phenomenon is unlikely to be limited to MM1 sCJD cases [24, 32].
Variability in sCJD patients The nature of peripheral tissues that accumulated infectivity in sCJD and vCJD cases were relatively similar. While the patterns of prion distribution and the infectivity levels observed in the peripheral tissues were relatively homogenous in the vCJD cases, particularly in lymphoid tissues, a high degree of variability was observed across the sCJD cases. Brain vacuolar lesion profiling and PrPres WB typing confirmed that the same prion strain was present in the brain and the peripheral tissues of all the sCJD affected patients. This rules out the hypothesis that prion strain(s) difference(s) (between individuals or in tissues from a same individual) could be responsible for the observed variability.
The comparison of prion distribution pattern in sCJD case 2 (20-months clinical phase duration) and sCJD cases 1, 3, 4 and 5 patients (1–4 months clinical phase duration) might suggest that, at first glance, a longer clinical phase duration is likely to be associated with a more widespread distribution of prions in the body tissues. However, even if our study represents an unprecedented effort for characterizing CJD agent distribution patterns in the organs of affected patients, the number of sCJD cases that we investigated is too limited to draw definitive conclusions. The characterisation of a larger cohort of sCJD patients will be necessary to establish the relationship that might exist between the distribution and/or infectivity levels in peripheral tissues and sCJD patient age at clinical onset and the duration of the clinical phase of the illness.
Iatrogenic transmission of CJD Several hundred cases of iatrogenic CJD transmission (iCJD) have been reported worldwide, the vast majority of which are likely to represent transmissions from sCJD patients [9]. The principal sources of these outbreaks were intramuscular injections with contaminated human pituitary-derived growth hormone (226 cases) and implantation of dura mater grafts (228 cases) derived from human cadavers with undiagnosed sCJD infections. A small number of cases were apparently also caused by neurosurgery using contaminated neurosurgical instruments and EEG electrodes (6 cases), transplantation of corneal grafts (2 cases) and intramuscular injections with human pituitary-derived gonadotrophic hormone (4 cases) [9].
These cases dramatically illustrate the high resistance of CJD prions to standard medical decontamination procedures and their particular abilities to bind to steel surgical instruments [19, 53]. Survival times in the individuals who were exposed to sCJD agent(s) by the peripheral route could be extremely long and variable; for instance, in patients that received intramuscular injection of contaminated human pituitary-derived growth hormone, the onset of clinical signs and symptoms of iatrogenic CJD could be observed between 4 and 42 years after the treatment [9].
In a context where sCJD infectivity is apparently limited to the CNS, the medical and surgical procedures responsible for iatrogenic transmission of the disease remain relatively limited, the overall risks for sCJD iatrogenic transmission are now considered to be remote since most of these established routes of transmission be avoided (e.g. the use of human pituitary-derived hormones and human dura mater grafts) [9].
Our detection of low levels of sCJD infectivity in non-CNS tissues such as lung, heart, muscle or even salivary gland was unexpected. We next reviewed the medical histories for the sCJD patients that we studied (Table 1), which revealed surgical procedures (s-CJD-2 and 4) and/or invasive medical examinations (polypectomy under colonoscopy sCJD-5) only few years before the clinical disease in these patients. Some years ago, the presence of infectivity in the plasma and the detection of abnormal prion protein in the urine of sCJD patients raised concerns about the risks of sCJD transmission by blood transfusion and plasma/urine-derived medical products [17, 38]. The detection, in our study, of infectivity in the bone marrow and the kidney of sCJD patients further reinforces these concerns.
Prion titres as measured by intracerebral inoculation in PrP over-expressing transgenic mice models can provide us with estimates of the relative infectivity levels present in the CNS and peripheral tissues of affected patients. This approach conforms to the current gold standard for quantification of prions. However, in a context where the amount of infectivity that would be necessary to transmit disease to another human remains unknown, the infectivity titre estimates (as established following intracerebral inoculation in a reporter animal model) cannot be used to directly infer transmission risks to patients. Other factors, including the various different potential exposure routes (subcutaneous, intramuscular, etc.) and the decontamination/sterilisation methodologies used on the surgical instruments /materials involved, will also influence the transmission risk analysis [1].
In many industrialized countries, reliable CJD passive surveillance programs have been established for decades. The apparently stable and low prevalence of sCJD cases in these countries bring some reassurance about the low numbers of iatrogenic CJD cases in this century [9], perhaps reflecting the variable and lower prion titres detected in the non-CNS tissues in this study.
Guidelines are in place to mitigate and control the risk of iatrogenic transmission of CJD in a healthcare setting [1, 56]. The procedures for cleaning and decontamination of surgical instruments and/or medical equipment now includes an assessment of their potential contamination by prions. However, prions are notoriously resistant to physico/chemical treatments and decontamination process that would be efficient on these agents remain generally inapplicable to some surgical and medical equipment [54], resulting in recommendations to destroy neurosurgical instruments that have been used on the brain of a patient with definite or probable CJD. Furthermore, the presence of dementia or an evolving neurodegenerative disorder in a patient undergoing medical or surgical procedures triggers the use of specific protocols designed to prevent the risk of potential transmission of CJD from the equipment used (surgical tools, endoscopes, etc.). The use of cells/tissues/organs and body fluids from these groups of patients for therapeutic purposes (blood donations, tissue grafts, etc.) is also restricted [1, 56].
When considering the risk of iatrogenic transmission of prion agents from CJD patients, one fundamental question is: How early before the occurrence of clinical signs and symptoms is prion infectivity likely to be present in peripheral solid tissues, blood and urine?
All the samples that we used to establish infectivity levels in tissues were collected post mortem in sCJD and vCJD patients at the terminal stage of the disease. There is clear evidence for the presence of infectivity in the blood and the peripheral tissues years before the clinical onset of vCJD in asymptomatic infected patients [3, 8, 16, 17]. However, it is uncertain that the infectivity levels and distribution in the post mortem peripheral tissues of sCJD and vCJD patients reflect the situation that could be observed at a preclinical or early clinical stage in the same patients. In the absence of tissue samples collected from asymptomatic sCJD patients we are totally lacking in data, therefore trying to elaborate further on this question would be totally speculative.
In several forms of genetic prion disease, for example those associated with the E200K PRNP mutation, the clinical disease manifestations, PrPres WB signature and tissue PrPres distributions are similar to that in sCJD [34, 48]. Despite the ethical issues it might raise, the longitudinal collection of blood samples and body fluids, for research purposes, in consenting patients belonging to families affected by these genetic forms of prion diseases (with confirmed mutations of the PRNP gene) may represent the only possibility to address this question.
In conclusion, the systematic surveillance of CJD and related epidemiological studies in many countries confirm the decline of cases of iatrogenic CJD due to recognised medical or surgical procedures, such as human dura mater graft surgery or treatment with human pituitary-derived growth hormone. However, they do not exclude the possibility that iatrogenic transmission could at least partly account for some sCJD cases observed in the population, particularly in localized geographic regions with evidence of CJD case clusters [50].
Many uncertainties remain on the early stages of the prion accumulation and infectivity in the peripheral tissues in patients infected with sCJD. However, the results of this study suggest that the iatrogenic transmission risks associated with sCJD peripheral tissues should not be disregarded.
Data and materials availability
All data are presented in the manuscript and the supplementary information. Human biological material used in the study was provided by the National CJD Research & Surveillance Unit Brain and Tissue Bank in Edinburgh, UK, which is part of the Edinburgh Brain and Tissue Bank supported by the Medical Research Council.
References
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