One of the world's main horticulture problems is the contamination of fruit trees with a variety of plant diseases, especially viral and pseudo-viral diseases. Due to the non-sexual propagation of the trees, these diseases have been transmitted to different parts of the world. The main aim of this study was to obtain a new effective method for virus elimination from almond cultivars, which was performed in two phases. In the first phase, we tested various almond cultivars with ELISA and RT-PCR. The results showed the infection of mother plantlets. So, three types of in vitro thermotherapy treatments were performed on infected plants to make them virus-free. The plantlets obtained from 0.5 mm meristem treated with the first type of thermotherapy (TH1: 8 h at 27 °C and 16 h at 38 °C for 18 days) showed the highest percentage of elimination of ApM, ACLS and TRS viruses. In the second phase, meristems were cultured on MS medium containing 0, 0.5, 1 and 2 mg/L 2,4-D with 1 mg/L TDZ and after two weeks, thermotherapy treatments were performed. The results showed, combining three methods of thermotherapy (TH1), meristem culture and somatic embryogenesis induction from meristem on MS medium supplemented with 0.5 mg/L 2,4-D and 1 mg/L TDZ is the most effective and safe technique for virus eradication without meristem size challenges. The samples that were diagnosed as virus-free were proliferated in temporary immersion bioreactor systems, and rooted to be used for later propagation and establishment of mother healthy orchards. With the increase in almond cultivation around the world, scientists are looking for factors that increase its yield. Therefore, the implementation of research programs in the field of health of commercial cultivars in each country is of great importance and leads to an increase in production per unit area. On the other hand, the yield in orchards with healthy cultivars is typically about 1.5–2 times more than control orchards2. Therefore, the use of efficient techniques such as shoot tip meristem culture, somatic embryogenesis and thermotherapy can be used to obtain virus-free plants7. Meristems are virus-free in most cases due to the lack of a vascular system. Studies have shown that culturing meristems of smaller sizes successfully results in virus-free plants12. In addition, high temperatures prevent virus replication by inhibiting virus transcription enzymes and reduce the transportation of virus towards the apical meristem13. Somatic embryogenesis eliminates the virus due to the lack of connection between the somatic embryo and the mother plant. In addition, somatic embryos are induced from non-vascular tissues, while viruses are mostly confined to vascular tissue, therefore, the chances of eliminating the virus increase14.However, it is important to have knowledge about the viral contaminants in almond trees, and the need to provide healthy vegetative materials for mother orchards. Various viruses have been reported in almonds, including prunus necrotic ringspot virus (PNRSV), apple chlorotic leaf spot virus (ACLSV), apple mosaic virus (ApMV), tomato ringspot virus (TRSV) and Mediterranean almond virusesFinding a proper technique in order to eradicate virus from almond trees is an important key to achieving healthy orchards and crops. Therefore, in the first phase of the present study, the effectiveness of thermotherapy treatments with meristem culture and in the second phase, for the first time, induction of somatic embryogenesis from meristem undergone thermotherapy was studied to investigate the role of these treatments in viral almond eradication. The presented results could develop the culture of virus-free germplasm of these trees as well as other fruit trees.15 containing 1 mg/L BAP, 0.5 mg/L GA3, 0.01 mg/L IBA and 30 g/l sucrose and 7 g/L plant-agar after disinfection with 70% alcohol for 1 min, then with sodium hypochlorite (2.5%) treatment for 10 min and nano-Silver (2.5%) for 10 min.Shoot samples were prepared from six local and commercial almond cultivars including Shahroodi, Shokofeh, Araz, Scandar, Nonpareil and Sahand from the almond germplasm collection of Horticultural Sciences Research Institute . The single lateral buds were transferred to the (MS)16 and (QL)17 and three types of growth regulators combination were studied to optimize the proliferation medium as follows Each medium contained 30 g/L sucrose and 7 g/L plant-agar. After pH adjustment on 5.8, the media were autoclaved for 15 min at 121 °C (1.2 kPa) and then the explants obtained from the establishment stage were cultured. The experiment was performed as factorial arrangement in completely randomized design with 4 replications. In a separate experiment, the best plant and the best medium were selected and the performance of the culture medium was evaluated as liquid (culturing in temporary immersion bioreactor system) and solid. Hence, the explants grown on MS medium with 1 mg/L BAP were sub-cultured to bioreactor container containing 330 ml of liquid MS medium supplemented with 1 mg/L BAP, 0.5 mg/L GA3, 0.01 mg/L IBA and 3% sucrose. Four explants with 1.5 – 2 cm length from lateral buds were cultured in each bioreactor container. The flow of the culture medium from the reservoir to the explants was regulated once every 24 h for 10 min. Cultures were incubated at a temperature of 25 ± 2 °C and 16 h light photoperiod at about 3000 lx. The solid medium was prepared with the same compounds as the liquid medium supplemented with 7 g/L plant-agar. Four explants with 1.5–2 cm length from lateral buds were cultured in each jar container. After 30 days, fresh weight (FW), dry weight (DW), shoot number (SN) and shoot length (SL) were measured. The experiment was performed in completely randomized design. The treatments were compared in 3 replications (3 jars or 3 bioreactor containers), which were incubated at a temperature of 25 ± 2 °C and 16 h light photoperiod at about 3000 lx.Before applying virus eradication treatments, in vitro cultured almond cultivars was evaluated by ELISA kit  for the presence of TRS virus. The plantlets were also tested for this virus by reverse transcription polymerase chain reaction (RT-PCR) assays. Total RNA was extracted from almond samples for RT-PCR and then synthesis of cDNA by reverse transcription and subsequent PCR was done by using the two-step kit .Before applying virus eradication treatments, we examined the contamination of mother plants using RT-PCR. The evaluated viruses included TRS, ACLS, and ApM. A proper sequence was selected to design primer pairs for each of the examined viruses from the previous reports Table .Table 2TFor specific amplification of the desired sequence, different annealing temperatures of the primer pairs were evaluated using temperature gradient. The temperature of 59 °C was finally used for all primer pairs. The small PCR products were loaded on the 1.5% agarose gels for detection.22. After completing the extraction process, concentration and quality of the extracted RNA were checked using a NanoDrop device and then by electrophoresis. Subsequently, about 4 µL of each extracted RNA sample was loaded on 1.5% agarose gel and after electrophoresis the resulting bands were evaluated. Based on the concentration (ng/µL) obtained from NanoDrop, the amount of the extracted compound containing 1 µg of RNA was taken and l µL Dnase enzyme plus l µL buffer was used to eliminate DNA.Extraction of RNA from plant leaves was performed according to the modified CTAB protocolThe successful synthesis of cDNA was examined with a proper primer pair from the housekeeping gene (a Rubisco subunit) by polymerase chain reaction. After observing the expected PCR product band on the electrophoresis gel, the manufactured cDNA was stored for later use in − 20 °C.After ensuring that the cDNA was constructed, the PCR reaction was performed using all virus specific primers for each sample of the constructed cDNA from mother plants. The PCR reaction product of each sample was loaded on the agarose gel and separated by electrophoresis. These diagnostic tests were also performed after the virus eradication operation to determine healthy plants. After virus-free operations and plantlets propagation, the percentage of virus-free plantlets detected by RT-PCR for each type of treatment was evaluated in a factorial arrangement in a completely randomized design with 3 replications.Two experiments were designed to optimize virus elimination by thermotherapy and shoot tip meristem culture. In the first experiment, three thermotherapy methods were applied on proliferated plantlets including TH1: 8 h at 27 °C and 16 h at 38 °C for 18 days, TH2: 10 days at 38 °C and TH3: 11 days at 38 °C; for gradual adaptation to this temperature, explants were first placed at a lower temperature (27 °C) and temperature was gradually increased (by around 3 °C increase per day). The experiment was performed as factorial arrangement in completely randomized design with 3 replications (petri dish) containing four 0.5 mm meristems. Meristem culturing was performed after thermotherapy . In the second experiment, meristems were separated from the explants undergone TH2. The primary separated apical meristem was approximately 5 mm with the lateral leaflets. Finally, the leaflets were removed and 0.5 and 1 mm meristems were cultured on the modified MS medium supplemented with 30 g/L sucrose, 7 g/L plant-agar, 0.1, 0.2 and 0.4 mg/L BAP and 0.1 mg/L IBA. The explants were incubated in a 24 ± 2 °C growth rooms with a 16 h light and 8 h of darkness photoperiod and light intensity of 2000 lx. There were 4 repetitions (petri dish) for each treatment; containing four meristems and the experiment was performed as factorial arrangement in completely randomized design. Following 30–35 days, the grown meristems were transferred to a new culture medium, and after sufficient growth, the virus tracking tests were applied.Meristems were cut to 0.5 and 1 mm sizes and cultured on the modified MS medium supplemented with 0, 0.5, 1 and 2 mg/L 2,4-Dichlorofenoxiacetic Acid , 1 mg/L thidiazuron (TDZ), 30 g/L sucrose and 7 g/L plant-agar. After two weeks of incubating at 24 ± 2 °C in a dark environment, they underwent two methods of thermotherapy  in a dark environment; for gradual adaptation to this temperature, explants were first placed at a lower temperature (27 °C) and temperature was gradually increased (by around 3 °C increase per day). There were three repetitions (petri dish) for each treatment, containing four meristems and the experiment was performed as factorial arrangement in completely randomized design. After thermotherapy, developed plantlets were propagated in temporary immersion bioreactor system containing liquid MS medium supplemented with 1 mg/L BAP, 0.5 mg/L GA3, 0.01 mg/L IBA and 3% sucrose.Plant growth regulator free MSPlant growth regulator free 1/2MSMS medium + IAA (1 mg/L)1/2MS medium + IAA (1 mg/L)MS medium + IAA (1 mg/L) + IBA (0.5 mg/L)1/2MS medium + IAA (1 mg/L) + IBA (0.5 mg/L)The explants were examined for ACLS, ApM and TRS viruses using molecular RT-PCR method. In addition, the serological ELISA method was carried out for TRS virus detection. Virus-free explants were amplified in temporary immersion bioreactor system supplemented with 1 mg/L BAP, 0.5 mg/L GA3, 0.01 mg/L IBA and 3% sucrose and then rooted in the half strength MS (1/2MS) medium supplemented with 1 mg/L IBA and 0.5 mg/L IAA. Proliferated shoots of 3 to 5 cm were used for rooting on MS or 1/2MS media supplemented by different concentrations of Indole-3-butyric acid (IBA) and Indole-3-acetic acid (IAA). The experiment was performed as factorial arrangement in a completely randomized design with 6 treatments and 4 replications. Explants were cultured in jam jars, with 3 explants in each, and were transferred to the photoperiod of 16 h light and 8 h darkness. Root number (RN) and root length (RL) were recorded after 6 weeks. Rooting treatments included:p < 0.05 or p < 0.01) was applied to establish the significance of the differences between each group, and the relevant graphs were drawn using Microsoft Excel. All data is displayed with standard error (± S.E).In the present study, as mentioned in the previous sections, seven experiments were performed, one in a completely randomized design and the other six in factorial arrangements in a completely randomized design. This study was done on 6 cultivars. The results of the interaction effects of 6 cultivars × different factors for each experiment are presented in the supplemental file and the interaction effects of 2 cultivars × different factors are presented as the main results. Statistical analysis of the measured characteristics was conducted using SAS (Version 9). Multivariate variance analysis was applied to determine the significance of differences between assays. Comparative mean LSD test  with different types of media . The results indicated that average number of shoots per explant for the 6 cultivars was 3.15, 1, and 0.78 on MS, QL, and WPM, respectively . According to the results of this study, number of shoots, fresh weight and dry weight as growth indices, in temporary immersion bioreactor system have increased significantly compared to solid culture medium  and there was no significant difference between the 0.5 and 1 mm meristem sizes in this respect. In terms of the percentage of plantlets produced from the determined embryogenic calluses, the highest performance was related to meristems with sizes 0.5 and 1 mm and concentration of 0.5 mg/L 2,4-D undergone TH1 for both cultivars  from the infected plum tree explants, in vitro thermotherapy was performed for one week at 38 °C with meristem tip culture3. In another study on apple cultivars, they examined the combined thermotherapy and cryotherapy effects. In this method, alternating temperatures of 38 °C (day) and 30 °C (night) were applied. Combining 4 weeks of thermotherapy with cryotherapy resulted in a high percentage of eradication32. Cryotherapy is a method to remove obligatory pathogens from meristem cells, which is a relative success. On the other hand, there is a possibility of long-term cryopreservation of pathogens in this method. Therefore, cryotherapy acts as a double-edged sword and thermotherapy is preferred34. Thermotherapy for more than 25 days caused the leaves to turn brown and the shoots to die. Culture of shoot tips along with thermotherapy (37 °C for 35 days) was necessary to regenerate virus free plants8. Increasing temperature and duration of thermotherapy has a positive correlation with virus eradication frequency. These two parameters vary depending on the type of virus and plant species and the virus-host combination. In fact, these two parameters should be optimized in such a way that it allows for the treated plant to survive and grow while inactivating the virus. Thermotherapy has several advantages over other methods such as cryotherapy, but many plants cannot tolerate high constant temperatures. Therefore, thermotherapy using alternating day/night temperature causes the growth and survival of the treated plant as well as the eradication of the virus35. In one study, thermotherapy was performed at 37–40 °C for 4 weeks and then apple meristems of different sizes were cultured. The size of the meristems affected their survival. Meristems of 0.6–0.7 mm had the highest percentage of establishment. MS medium with BA (1 mg/L), IBA (0.05 mg/L) and GA3 (0.1 mg/L) was the best culture medium for meristem establishment36. In another study, sweet cherry meristem explants (less than 1 mm) were isolated from the tip of the shoot and cultured on MS medium. The results showed that there was no significant difference between cultivars in terms of survival index as well as proliferation rate and shoot length37. In general, the size of the shoot tip has a positive correlation with survival and successful production of plantlets and a negative correlation with the frequency of virus eradication35.Generally, in vitro virus eradication based on meristem culture is common and in addition, the other treatments including thermotherapy, chemotherapy, electrotherapy and cryotherapy can be used. During each of these treatments, the explants are subjected to a number of stresses that can lead to low survival rates, growth inhibition, stunted growth, or abnormal morphology. The sensitivity of genotypes to the treatments of these processes is very different, and the degradation degree largely depends on the plant physiological conditions. The exposure time to each of the processes affects the damage extent in treatments38. Detecting fruit tree viruses has been challenging for a long time; there are two reasons for this: in the first case, the viral titer is often low, and in the second case, the viral is unevenly distributed among the tissues and branches of different fruit trees. RT-PCR consists of two steps involving viral RNA transcription using total extracted RNA and PCR amplification from viral cDNA39. The identification of plant viruses is of great importance as a prelude to successful management of a viral disease. Recent advances in molecular biology have led to the development of new, sensitive, and effective diagnostic methods. Current trends in plant virus detection tools have provided equipment that do not reduce sensitivity and reproducibility, such as RT-PCR40.According to Table Prunus dulcis. For example, cultivation of anthers in P medium, cultivation of different parts of immature cotyledons  on MS medium containing 0.2 mg/L BAP and 0.01 mg/L IBA with different concentrations of TDZ  plays an important role in initiating the induction of somatic embryogenesis in the proximal and median parts of the cotyledons42. In another species of Prunus (P. cerasus), immature cotyledons were isolated after pollination and cultured on MS medium and somatic embryogenesis was reported to occur mainly when using 2,4-D in addition to kinetin43.Somatic embryogenesis induction was performed from different tissues of 44. Also, virus-free garlic plants were regenerated through somatic embryogenesis from basal parts of cloves of two Croatian garlic ecotypes on MS medium containing 0.1 mg/L 2,4-D. The results showed that the elimination of onion yellow dwarf virus (OYDV), leek yellow stripe virus (LYSV) and garlic common latent virus (GCLV) was as successful as other methods45. Somatic embryogenesis of tepal flower tissue in Hippeastrum hybridum was performed on MS medium containing 0.5 mg/L 2,4-D and 1 mg/L TDZ. Plantlets from somatic embryos were successfully free of Cucumber mosaic virus and Hippeastrum mosaic virus7. According to studies in India, elimination of Piper yellow mottle virus (PYMoV) increased when cyclic somatic embryos were pretreated with ribavirin before induction46.Somatic embryogenesis is performed to produce virus-free plantlets from different plant tissues, for example, somatic embryogenesis from anthers was the most promising approach to producing arabis mosaic virus-free grape plantsAmong thermotherapy treatments for virus eradication, storage of plantlets 8 h at 27 °C and 16 h at 38 °C for 18 days was the best treatment considering the highest regeneration percentage of survived meristems. The hormonal combination containing 0.4 mg/L BAP was the best treatment used for establishment of meristem. Plantlets obtained from 0.5 mm meristems, which underwent thermotherapy treatment for 18 days at 8 °C and 16 h at 38 °C (TH1), showed the highest percentage of virus elimination for all three viruses  in different cultivars.Combining three methods of thermotherapy (TH1), meristem culture and somatic embryogenesis induction from meristem on MS medium supplemented with 0.5 mg/L 2,4-D and 1 mg/L TDZ is the most effective technique for virus eradication without meristem size challenges.Virus-free explants were amplified in temporary immersion bioreactor system supplemented with 1 mg/L BAP, 0.5 mg/L GA3, 0.01 mg/L IBA and 3% sucrose and then rooted in the half strength MS (1/2MS) medium supplemented with 1 mg/L IBA and 0.5 mg/L IAA.Supplementary Information.


Previous research has emphasized the importance of therapists giving Routine Outcome Monitoring (ROM) feedback to their patients. It has been shown that several factors influence therapists’ tendency to provide ROM feedback to their patients.In this qualitative study, using a semi-structured interview followed by thematic analysis using Atlas.ti, we focused on experiences of therapists and patients with a disorder specific ROM instrument: the Borderline Personality Disorder Severity Index-IV (BPDSI-IV). Ten patients with a borderline personality disorder who had been in Mentalization Based Treatment (MBT) and ten MBT-therapists treating patients with a borderline personality disorder were interviewed. Qualitative analysis revealed that patients experienced benefits of ROM using the BPDSI-IV. Patients gained more insight in and recognition of their borderline personality disorder symptoms. They also felt more understood by the therapist because they got an opportunity to explain their symptoms in a different way than in a regular therapy session. Therapists shared they didn’t always use all the ROM outcomes as serious feedback for adjusting treatment. They preferred to use the BPDSI-IV over the other ROM instruments, because the BPDSI-IV is disorder specific, which gives insight into the treatment course of the patient.Experiences of both patients and therapists with the BPDSI-IV were positive. It seems to be valuable and promising for healthcare institutions to evaluate treatment with a disorder specific ROM instrument.The online version contains supplementary material available at 10.1186/s12888-022-04104-w. From all patients in therapy, only 50%-75% benefits from treatment . It is uThere are several possible reasons for not ending therapy while no progress is being made: i.e. patients are afraid to deteriorate; patients are feeling supported by their therapist; therapists do not want to disappoint their patients; therapists want to achieve the best result as possible; therapists do not want to admit that treatment is not bringing enough positive change .The accuracy of therapists’ judgment for predicting if patients will deteriorate in treatment seems to be less accurate than the accuracy of a routine outcome instrument. In a study of Hannan et al.  therapisTimely ending unsuccessful treatment will reduce waiting lists and high costs and gives patients a chance to enroll in a potentially more effective treatment . To helpFeedback on treatment progress is most effective when it is given to both therapist and patient . UnfortuPrevious research about therapists’ and patients’ experiences with ROM was mainly focused on self-report questionnaires; questionnaires which are not disorder specific; and ROM experiences of therapists. The listed criticism was also reported among clients with severe BPD and their therapists who found the standard generic ‘Brief Symptom Inventory’  clinicaln = 10) and therapists (n = 10) from the Mentalization Based Treatment (MBT) program at ‘de Viersprong’, Amsterdam. De Viersprong is a Dutch institute specialized in psychotherapy for personality disorders.Participants were patients . DSM-IV diagnoses were assessed by trained clinicians using Structured Clinical Interviews for DSM-IV axis I , 15 and n = 1), master-level psychologists (n = 2), therapists with a post-master degree in Healthcare psychology (GZ-psycholoog) (n = 4), psychotherapists (n = 2) and (post-master) clinical psychologists . Eight were female. Therapist selection was representative in disciplines, age and gender.All therapists were extensively trained in MBT and working under the MBT Quality system . They han = 5), 2) three day day-treatment (n = 1) and 3) five day day-program (n = 4). The intensive outpatient treatment included two times a week mentalizing group psychotherapy, weekly individual psychotherapy, patients received socio-therapeutic consults when indicated and medication consults on request of the team and patient. The three day day-treatment included three times a week mentalizing group psychotherapy, weekly individual psychotherapy, weekly art therapy, weekly mentalizing group therapy, weekly writing therapy, patients received socio-therapeutic consults when indicated and medication consults on request of the team and patient. The program ended with a social hour and community meeting. The five day day-treatment included five times a week mentalizing group psychotherapy, weekly individual psychotherapy, twice a week art therapy, weekly mentalizing group therapy, weekly writing therapy, patients received socio-therapeutic consults when indicated and medication consults on request of the team and patient. The program ended with a social hour and community meeting.The MBT treatment program consisted of introductory groups (10–12 sessions) , treatmeROM at de Viersprong was used to measure treatment outcomes. Measures were taken every three months by a trained treatment-independent research assistant. Patients and therapists were provided a written ROM-report with baseline, next to last assessment, and last assessment outcomes. Therapists were expected to discuss the outcomes with patients and use the outcomes to guide therapy course and duration. All Therapists were trained in interpreting and using ROM-feedback as a standard procedure in their introductory period.Measures used at each assessment were the Brief Symptom Inventory (BSI) , the SevThe BSI  is a selThe SIPP-sf  is a selThe PDS –20 is a The BPDSI-IV  is a sem‘Did ROM give you a better insight into the treatment course of your patient? (YES) Can you tell us how you got that insight? (If applicable) Which lists gave you this insight?’ The patient-interview contained five topics in the context of ROM: general questions, relation between therapist and patients, feedback, explanation of therapist about ROM and BPDSI-IV. An example of a question from the patient-interview is: ‘Did the feedback from the ROM provide you with more insight into your symptoms? (YES) What kind of insights did you get? Based on what did you get these insights?’ The full interviews are presented in Additional file A semi-structured interview was designed for this study to investigate the experience of therapists and patients with ROM. The therapist-interview contained five topics in the context of ROM: relation between therapist and patient, feedback to patient, ROM and BPDSI-IV in general. An example of a question from the therapist-interview is: n = 11), did not pick up the phone (n = 20) or the phone number didn’t exist anymore (n = 6), the next patient on the list was called. There were three lists of patients, one for every of the three different MBT programs: intensive outpatient treatment (n = 34), three day day-treatment (n = 10) and five day day-treatment program (n = 34). When patients wanted to participate in the study an appointment was made. Appointments were made with twelve patients, there were two no shows. Five patients of the intensive outpatient treatment, one patient of the three day day-treatment and four patients of the five day day-treatment did participate. Participants were given the option to make a face-to-face appointment at de Viersprong (n = 4) or an appointment by telephone (n = 6). Participants received general information about the nature of the study and the informed consent letter by e-mail. Appointments did not take longer than one hour and participants received 12.50 euros for their participation. Interviews were audio-recorded and transcribed verbatim.Patients who had been in MBT treatment were recruited by phone. When patients did not want to participate (n = 10). Interviews were audio-recorded and transcribed verbatim.A short introduction about the nature of the study was given to therapists during a MBT meeting at de Viersprong Amsterdam. It was explained that the study goal was to get therapists’ and patients’ experiences to optimize ROM in MBT. All MBT-therapists received an information letter about the study. Afterwards the therapists received an email with a couple of optional dates for an appointment. All therapists entered the study after giving written consent. Appointments did not take longer than one hour and therapists received 12.50 euros for their participation. Appointments with therapists were face-to-face at de Viersprong .The second author (SC) analyzed the data, based on the semi-structured interviews, thematically. The patient-interviews and the therapist-interviews were analyzed separately. SC read and re-read the transcripts of the interviews to gain familiarity and to gain an overall impression of the collected data. Relevant fragments that seemed to be meaningful were coded and organized in (sub)categories based on the interview topics. Some codes were not distinctive and were merged. SC combined the categories in overarching topics: experiences of therapists and patients with the BPDSI-IV, experiences of therapists and patients with ROM in general and importance of emphasizing ROM culture in organization. A mixed top-down/bottom-up procedure was used to determine the themes. Analyses were conducted in Atlas.ti.Table Well I think it is very good but what I find difficult and certainly found it, especially in the beginning, super confrontational with someone you don't know very well, with whom you are not really going to build an attachment relationship because it is not one of the therapists, to talk about it in that way. Yep, that's just really scary. On the other hand, I think it helped. If I now feel insecure or anxious, it is very difficult for me to use my social environment for this. And my social environment are people that I have selected myself, where I feel confident or not myself. But then when I find that difficult, my mistrust increases and my confidence decreases and I find it more difficult to be vulnerable. Suppose I only had the therapists then I could still think very much 'yes, they are specialists, they will understand me’. And although the person who takes the ROM is of course not just someone off the street, you are kind of forced to talk about it. So I feel it is also a kind of exposure or something and I like that but I also found it difficult.”“The results allowed me to take more account of myself. For example, ‘why is it that my anxiety symptoms have increased?’, then I could reflect on these feelings. That's what I learned here: standing still. Normally, I haven't done that in years. I'm someone who walks away from my feelings. Whatever the outcome was, I always tried to talk to the practitioner about it, about what I had to do, how I can improve.”“Nine out of ten patients reported at least one benefit of the repeated assessments with the BPDSI. Examples of the various benefits that patients mentioned were: they liked that it specifically measures borderline symptoms (in contrast to the other ROM instruments); that the BPDSI was repeatedly used clarified whether therapy was making a difference; it is exposure to discussing their feelings with someone other than the therapist; that it made them more aware of their symptoms; that it made the symptoms more clear for them; and that it gave them the feeling that the disorder became more clear to the therapist.In any case, I prefer a conversation more than filling out a questionnaire, it is more personal. Questionnaires are generally the same for everyone. In the conversation you can give your own explanation more, more specifically tell something detailed or something. You can go deeper into it than with a questionnaire. A questionnaire does not really suggest an addition or something. I can never make an addition or add an explanation, I can do that in an interview.”“A patient mentioned that from the results of the BPDSI-IV it became clear to the therapist that the patient had self-injured himself, while they had not talked about this subject in therapy. One patient experienced the interview as more personal than filling in a questionnaire (on the computer), because it was possible to explain her answers (note that the BPDSI is a semi-structured interview).“Again the interview part because that was what really made me think. You can simply click away those other questionnaires. And you know that at some point if you walk along in psychiatry as a client for a while, then you have had the questionnaires so many times that you can dream them. And actually you think at that moment, ‘it says last week or last three months or last six months’, and then you try to think a bit about it, but you don’t do that very much. While in that interview you do that because it has a completely different approach. And you are much more forced to consciously think about the past week or the past three months.”Also another patient seems to prefer the approach of the BPDSI interview above the questionnaires on the computer. The interview made her think more consciously about the symptoms she had experienced.“It is a kind of reality check with yourself. You have to give an hour and a half very conscious answers about a recent period and how often something happened. And you find out that one thing still happens more often than you like and the other has actually been reduced a lot. As a result, during the course of the treatment, you will also increasingly see what your own progress is. And that is very motivating to continue with the treatment.”Another patient mentioned that following his own treatment course with the BPDSI, motivated him to go on with treatment.Another benefit that was mentioned was that the results made it easier to explain the disorder to loved ones.“I can imagine that for other people such insight can also be important for the family, to explain that the therapy goes in a certain way, and I have decreased on this and I have just gotten better on that.”To summarize, the above patients gained insight because of the BPDSI-IV.Most of the therapists mentioned the disorder specificity as a benefit of the BPDSI-IV; it measured what they were treating the patients for. A therapist mentioned that the subscales of the BPDSI-IV made the symptoms of the disorder more insightful and it made it possible to see changes in treatment course.“And then at least you get a little more insight into: ‘hey, what about the different domains, and where are the biggest problems, and what about the emptiness, what about the relationship, what about..?’ ““So then yes, the BPDSI is what I like to look at, because then you have those subscales matching the BPD features. That those features of the borderline personality disorder are set out over different time periods. Yes, I find that is helpful in the light of: ‘where do I stand regarding my goals and the patient regarding his goals and do I recognize this in the subscales?’”Another therapist mentioned that the BPDSI-IV made it possible to classify and that the clear cut-off point made it possible to see if a patient still met the criteria of BPD.“it does make it very specific, the results on symptom level”.Another therapist: “Sometimes you really get tired of it. But after a while you think yes, you know this for yourself.”Most experiences of patients with the BPDSI-IV were positive, but seven out of ten also mentioned some disadvantages or recommendations. All but one of these seven patients also mentioned advantages or mentioned that the disadvantages did not outweigh the benefits. Two patients mentioned that the questions of the interview can be hard or difficult i.e. because it was hard to recall how many times things happened and another patient mentioned that sometimes she was done answering questions during the interview, but nevertheless the interview was an important part of the treatment for these three patients.“The only negative side is more tension for the patient, as a kind of exam; ‘Did I do it well or not?’ More the excitement of ‘what do I hear, how do I do.’ But I don't think that outweighs the image you get about yourself.”One patient felt as tense as before an exam, however, this did not seem to be as important as the insight the patient gained of himself.Maybe there are also questions between which you normally do not bother and that it gives you ideas or something, that could be. I would say [for] myself, when I am in a very difficult period and there will be a certain question that I had not thought about myself, then it could also give me ideas.”“The same patient also mentioned that he would like to receive the results consequent on paper. Another patient mentioned that some questions about symptoms, which the patient did not recognize at the moment, could lead to certain ideas.The same patient suggested using the interview questions in therapy sessions so they have more time to reflect on the issue..“I get in a mood, of course, that makes me remember things that are not nice, and of course it makes me sad or anxious. But I don't know if it is negative. So it is awareness.”It was also mentioned that it was confronting to undergo the BPDSI-IV interview with an interviewer which the patient did not know well and without building an attachment relationship. The same patient however explained that you could see this as ‘exposure’, which was a positive experience. One patient mentioned that the questions about addiction and using medication, fears, mood changes and sadness, brought back memories which made her sad and anxious. This patient was wondering if this is positive or negative because it also made her aware of past and current symptoms"Remove the pieces that do not suit me and spend more time on pieces that are more important to me."The same patient mentioned that a lot of the questions asked of her were no longer relevant which she referred to as a waste of time. For a long time, she was not cutting herself anymore but still it was asked in the interview. She recommended removing irrelevant questions and allocating more time to subjects that are more important to her..“Well, the report sometimes may be a bit more extensive. That there is a little more explanation about what exactly the symptoms are. Because then you see, for example, decrease on relationships or on emptiness, but I would like to add a little more words, also for the client. If the client can do something with it. But then you are mainly talking about the report, but I could suggest some improvement there.”Two therapists’ said they would like to see a more expanded report of the results of the BPDSI-IV, i.e. with more explanation about the symptoms or a verbatim of the interview“It is quite difficult to administer, you have to do an interview. I would really like it if there was a self-report list. Which requires less work, so that it could simply be used much more. Not only here but also elsewhere. Because I think the work where I was before, there was a lot of borderline. But you weren’t just going to do the BPDSI, because I also think that you already suggest a lot. Then you already suggest that someone has borderline. That is possible here because everyone has it when they come here, but in other settings that is much more difficult. So then I would really like it if there was another way.”One therapist mentioned the effort of the patient and interviewer as a disadvantage. Suggested was to take the interview only once every six months instead of once every three months and adding a BPD self-report form in the meantime. Another therapist also mentioned the use of a BPD self-report form which would take less time and would be more easy to use in treatment centers which are not focused on only BPD.“There was a patient who really had questions like: what is that ‘emptiness’? Then I really had discussions with her, and at a certain point I just took the BPSDI and went to see it with her, and I noticed that these are also quite literal questions about how it is described in the DSM. I think that is a disadvantage, it gives a lot of noise I think. Because it remains so abstract, for example emptiness.”One therapist mentioned the risk of using a semi-structured interview causing important things possibly to be missed because questions can be steered. Another therapist mentioned that the BPDSI-IV is quite literary based on the DSM, the result can be that the questions of the BPDSI-IV can be abstract to the patient, which could cause errors.‘We already know that we only treat people with borderline. I would like to know more; What do the symptoms look like? How intensive are they? What is the extent of suffering? Has it changed?’Another therapist mentioned that he would like to see more specific description of symptoms accompanying the subscales describing the nature, the frequency and the severity of the borderline symptoms at the time of the measurement.“Clients really can get a terrible tendency: 'yes, but I have to go below 15 because then I will be cured’. That is very sad when the next time is 17, then you have it again. They also become very anxious about it: 'I am now at 14, I have to get out of treatment now because then I no longer have borderline'. So I would like to have it a little less absolute. Also because it can still fluctuate strongly.”One therapist questioned the validity of the BPDSI-IV. Two therapists wanted the cut-off point to be less absolute.“So you measure the outcomes, the consequences of the personality problem, but you don't measure the personality problem in itself. So that's a disadvantage. But I understand very well that it goes like this. We also do a SCID in the intake here. You indicate someone here for treatment based on their symptoms. So I also understand that you measure it that way, but that is of course really a shortcoming. That you don't, understand how someone is as a person, and how things are going differently. You only measure the characteristics.”It was also mentioned that the BPDSI-IV only measures the DSM- personality characteristics and that it does not measure the level of personality functioning.I work in education, I teach, but I also want there to be a learning track and that we work towards something and that you can hold on to something that you can test. Yes I don't know. For me it makes perfect sense in the learning process that you test and monitor. And that you report results and you look back on it, I don't know. In my head that cannot exist without each other.”““I would prefer to always be in therapy, that is just very safe and you have a place where you can discuss everything. In the beginning it is very difficult to find that trust. And once you have that trust, it is very difficult to let it go and do it by yourself. I think it is very nice for both the therapist and the client that you have that ROM to say: ‘Yes, it is going well, so you can do it’. The moment you didn't have the ROM, it might just be based on the conversations. Now you really review exactly what happened in the past three months.”ROM feedback was usually given to patients during evaluations in which treatment plans were being discussed. Some therapists’ gave ROM feedback during an individual session . Patients mentioned that ROM feedback was used to make decisions in treatment, like formulating treatment goals or transition to aftercare and to monitor the treatment course.One patient describes it as “A piece of awareness. You are by no means aware of everything if it is not mentioned; that something goes better or worse.”Most participants gained insight in their disorder because of the ROM feedback. It gave insight into their own suicidality, the treatment course, the kind and the severity of the symptoms.Not all participants got a better insight in or monitoring of their symptoms through ROM, for example one participant mentioned a lack of confidence in ROM and another participant had a comorbid disorder which made ROM confusing.“Of course, a lot of questions are asked during ROM. Questions that may not normally surface during a conversation. So that you address other topics than you would just have in a conversation.”Four patients felt the therapists got a better understanding of the patient because of ROM. This was mentioned by two patients who found it hard to explain emotions or symptoms, one patient who had varying ROM results which was discussed in treatment, and one patient who felt that ROM brought up subjects which aren’t discussed in therapy sessions.There is a perverse incentive behind ROM.”” A therapist described ROM as: "ROM is actually just the figure with which you say: ‘where are we actually regarding this goal’. It is also very much about what we have actually achieved here in the recent period.”ROM was described by one therapist as initiated by and as an obligation of the insurance company. Money is invested in ROM but could also be spent on better care. However, that did not seem to be the dominant picture of ROM among the participating therapists.'quick screener' for present psychiatric symptoms, which can be discussed with the patient. ROM is seen as an addition to the previously formulated treatment goals and measuring every three months ensures that there are differences in symptoms over time.Another positive side effect of the ROM is described as: ‘An instrument that objectively goes through all those complaints and scales, it is an addition to your clinical view’.Another therapist gave the following example: “You work here in attachment relationships where transference plays a major role and your view can be clouded. If all goes well, this kind of measurement provides you with a clear view.”ROM was described as a It was experienced as pleasant that someone from outside the team, the test psychologist, was engaged. This, because the test psychologist is not directly involved in the dynamic of the patient and therapist. In addition, the patient is involved in the treatment by ROM.“For example, sometimes you come by a client whose ROM outcomes deteriorate greatly in the first few months, but sometimes that is not a bad sign at all. Because someone starts to feel more and think about these feelings more. In that case it is true the symptoms increase but then it would be misinterpreting ROM when one would say it is not going well. So in that sense I think the displayed outcomes are on a superficial level. So I just think ROM is not enough to say anything about the treatment result because my clinical view is also absolutely essential.”Therapists’ however also were critical about ROM and do question the reliability of ROM. It was frequently mentioned that ROM couldn’t be seen separately from clinical observations by the therapist. It was also mentioned that ROM is a snapshot, depending on situational circumstances.Another therapist: ‘Many of those questionnaires are self-report lists and they are not reliable in people with borderline. Some people can really display something quite well about how things are going now or how things have been going over the past period. Some people can't. The weaker people are structured, the more distortions there are sometimes. I think that is really a problem with these measurements.’ROM was described as expensive and time-intensive. At de Viersprong, four ROM instruments were taken, while one is the most used in therapy, namely the BPDSI-IV. A therapist wonders whether the cost benefits of the ROM would be positive.So then the experience of the patient becomes a piece of paper. The experience is externalized and the patient has to read on a piece of paper whether he is doing better and I find that very strange, I find that surprising. Because it assumes that we can measure it very well and that we therefore assume that it is a result of treatment.”” One therapist mentioned that there might be a learning effect among patients in answering ROM questionnaires, a coloring of answers due to not wanting to complete the treatment or, on the contrary, giving ‘better’ answers than the patient actually feels, or choosing a good day for filling out ROM. Patients may see the ROM as a school report or experience the feeling of having to perform. Therapists wonder if you can measure the effect of the treatment based on symptoms.Other shortcomings mentioned are possible bias, too many confounders, and a number of organizational shortcomings such as the planning of ROM.Therapists have different experiences with education about ROM. A therapist mentioned he hadn’t had any education about ROM and another therapist couldn’t remember having received education about ROM. Another therapist mentioned that therapists have undoubtedly been given information but was unable to remember this well. Three other participants also mentioned that education about ROM was a long time ago. Four participants mentioned that they had received an explanation about ROM from the test psychologist during their introduction period.Because now we do it a bit because it has to be done, and I can give it a twist, but that is really in my own way. Everyone would have a different way, so that doesn't seem very good to me either.”“It is remarkable that more than half of the therapists mentioned that they mainly used the BPDSI-IV in treatment and that they choose to not use the other lists because of various reasons. For example, a therapists mentioned that the PDS is too sensitive; two therapists mentioned that the PDS was not applicable for the patients; another therapist mentioned that the BSI always yielded high scores; and four therapists mentioned that they did not use  the instruments optimally due to a lack of knowledge. Examples of a lack of knowledge are therapists who are not sure how to explain or provide feedback about the lists, just recently found out that the BPDSI-IV is an interview, do not know how to integrate the lists together and neither how to integrate the lists in therapy, or are not familiar with the content of the lists. Therapists would like to receive more information about ROM, so they can use the ROM more optimally. There also seemed to be a need for knowledge about the developments of ROM with regard to the insurance companies, so they can explain this to the patient. Therapists were thinking about education about ROM in different types; brochures that are easy to copy, a refresher course every now and then, but also feedback about the aggregated ROM results of the department. A therapist also expressed a need for a guideline for feedback and interpretation of ROM:"I think when you can see what it gives you it is helping, but if you can’t then it becomes a [sigh] you know, then it just becomes something you have to do extra and you would think something like: ‘I am already telling to my therapist how it goes?’ And if you don't make that clear, it just becomes an annoying list. So yes, that is not actually my opinion, I think it can really have an added value, but you would also need a mutual culture to consider it in that way and the substantive added value is also visible.”A good implementation of ROM was seen as important. On the one hand to motivate patients for ROM, on the other hand to make therapists feel familiar with ROM.Another therapist mentioned the importance of training new employees in ROM in order to emphasize the importance of ROM in the organization.In this study we found that in general experiences of patients and therapists whit the BPDSI-IV were positive and the use of a disorder specific ROM instrument seems promising and valuable.As was found in other studies –23, therPatients and therapists seemed to prefer the BPDSI-IV above the other self-report ROM instruments. Patients mainly because it reflected their symptoms best, for therapists this was an important argument as well, but another reason was they did not know how to interpret the other instruments. This is remarkable since guidelines and knowledge about the use of ROM was available at de Viersprong; there were ROM workshops, there were flashcards about ROM, and the tests psychologists are there for uncertainties about ROM. Besides the possibilities about gaining information about ROM, the organization attempted to implement a strong ROM-culture; patients received ROM every three months via e-mail, patients were able to sign themselves up for the BPDSI-IV interview, therapists received monthly reminders via e-mail, ROM was part of the training program of new therapists, and it was part of the Quality System of MBT. Despite the many given possibilities for effective use of ROM, therapists do not seem to recognize a strong ROM-culture.A strong culture in using quality systems in organizations is important as it decreases the variation between therapists in use . DespiteEven in a ‘ROM-valuing organizational context’ ‘the doctor knows best principle’, mistrust pertaining to the incorrect use of ROM-data by insurance agencies or the organization, and the idea of patients not being capable of reporting their symptoms objectively and to the full extent runs freely among therapists. The contrast with patients experiencing ROM as helpful for self-esteem, treatment monitoring, and enhancing and shortening the treatment process is disturbing and in contrast with scientific outcome –22. The One needs to be careful with generalizing the results of this study to other patients and therapists. Limitations of this study are that both groups were of a small sample and both groups were part of one treatment center and of one specific treatment program (MBT). However, Guest, Bunch and Johnson  researchThe present study was conducted among patients who received MBT and among MBT-therapists. As type of therapy might be of influence, future studies should study patients’ and therapists’ views of different forms of psychotherapy.Although the patient and therapist samples where representative for both groups within the Viersprong, there where few men in both samples. Future research can shed light on the question whether there is any gender difference in the subjective experiences with Routine Outcome Monitoring. Other limitations of this study are that there was no member-check and analyses weren’t double classified by a second rater, so it wasn’t possible to calculate the inter-coder agreement. To limit this negative effect the coder was well supervised by the first author, hence the coding was not a pure individual-subjective activity. The fact that the study was unfunded limited our possibility to employ multiple coders.Since the BPDSI-IV needs training for interviewers and takes approximately 45–60 min per interview, future studies could compare the BPDSI-IV to other BPD-specific or a more generic Personality symptom self-report questionnaire such as the Severity Indices of Personality Problems (SIPP-118)  to invesAlthough the aim of the present study was to investigate how a BPD-specific outcome measure was evaluated by stakeholders, also in comparison to a generic psychopathological symptom outcome, the study did not address how an outcome assessing functioning would be valued. An important topic for future studies is to investigate how an outcome assessing functioning is evaluated, also in comparison to a measure assessing disorder-specific symptoms.Organizations could improve or extend their ROM-culture not only focusing on a facilitated ROM-process, but also to train and inform therapists about how to give ROM feedback, how to interpret ROM, how to combine ROM with clinical impressions (which indeed are important) and how ROM is exactly used within the organization and in relationship to insurance agencies. It also seems to be important to support therapist self-esteem and stimulate them to discuss ROM-output in their professional team and peer-supervision to overcome the (sometimes difficult to bear) idea the therapist did not improve the patient and open up the option a treatment does not fit, or the therapist might not fit the patient or the treatment. Mental health care can profit from a more open culture where making mistakes, or not being a good therapist (in a specific context) is an option. This will increase learning possibilities and expectedly decrease the number of unsuccessful treatments.As was found in this study , the preAdditional file 1.Interview patients.Additional file 2.Interview therapists..


To assess and compare clinical outcomes and costs, to the Italian healthcare system, of three therapeutic options approved in the management of adult patients with gastro-enteropancreatic neuroendocrine tumours (GEP-NETs).We compared the efficacy, safety, and costs of [177Lu]Lu-DOTA-TATE, everolimus (both originator and generic products), and sunitinib in patients with advanced GEP-NETs (NET G1 and G2) that had progressed following treatment with somatostatin analogs (SSAs). A cost-consequence model was developed and validated by a panel of clinical experts from three NET reference centres in Italy. The clinical outcomes included in the model were median progression-free survival and the incidence of grade 3 or 4 adverse events (AEs), as reported in pivotal clinical trials. The costs for acquisition and administration of each treatment, and of managing AEs, were calculated from the perspective of the Italian national health service. Treatment costs per progression-free month were calculated separately for patients with NETs of pancreatic  and gastrointestinal  origin.In patients with PanNETs, total costs per progression-free month were €2989 for [177Lu]Lu-DOTA-TATE, €4975 for originator everolimus, €3472 for generic everolimus, and €5337 for sunitinib. In patients with GI-NETs, total costs per progression-free month were €3189 for [177Lu]Lu-DOTA-TATE, €4990 for originator everolimus, and €3483 for generic everolimus.[177Lu]Lu-DOTA-TATE was associated with lower costs per progression-free month versus relevant treatment options in patients with GI-NETs or PanNETs , although acquisition and administration costs are higher. These findings provide further economic arguments in the overall context of treatment decision-making.The online version contains supplementary material available at 10.1007/s00259-021-05656-x. Neuroendocrine neoplasms (NENs), better known as neuroendocrine tumours (NETs), arise from the diffuse neuroendocrine system, which is distributed throughout the human body. Thus, NETs can originate in any part of the body, but most commonly arise in the gastro-enteropancreatic (GEP) tract. Indeed, GEP-NETs accounted for 3.56 cases per 100 000 in the Surveillance, Epidemiology, and End Results (SEER) 18 registry . In ItalHistologically, NENs can be divided into two main groups: well-differentiated tumours (NETs) and poorly differentiated neuroendocrine carcinomas (NECs). Additionally, NETs can be divided into four categories  depending on morphology and proliferation index (Ki-67); each has distinct clinical and biomolecular features and prognostic features in terms of survival .Early-stage GEP-NETs are often asymptomatic, or present with poorly defined symptoms . ConsequP < 0.001). In addition, the estimated risk of death was 60% lower with [177Lu]Lu-DOTA-TATE versus octreotide LAR  Lu-DOTA-TATE significantly lowered the risk of disease progression in patients with inoperable, progressive midgut NETs . At the = 0.004) . SubsequIn the absence of relevant data from randomised trials, the optimum sequence for the use of systemic treatments for GEP-NETs is unknown. However, one meta-analysis submitted in conjunction with a cost-effectiveness model to the National Centre for Health and Care Excellence (NICE) in the UK suggested a survival benefit of [177Lu]Lu-DOTA-TATE compared with current care . NICE prIn Italy and other countries, there is a need for a simpler model that is based on published PFS data and includes the costs of circumventing the unfavourable impact of prolonged OS. Moreover, research into biological and clinical predictors of response is still ongoing, and the profile of patients most likely to respond to each option is therefore unknown. In this context, a pharmacoeconomic model, designed to assess and compare the clinical outcomes and costs associated with competing therapeutic options, may be a useful aid to decision-making based on survival and cost aspects.Accordingly, we developed a specific novel cost-consequence model to evaluate treatment options, in clinical and economic terms, for Italian patients with advanced GEP-NETs .A cost-consequence model was designed to compare the clinical outcomes (efficacy and safety) and costs associated with three approved treatment alternatives  for patients with advanced GEP-NETs  in Italy. The main metric of this study was the cost of each treatment option per progression-free month, taking into account the costs of drug acquisition, administration, and toxicity.Median PFS data for patients with advanced (unresectable or metastatic) PanNETs or GI-NETs were taken from the official summaries of product characteristics (SmPCs) , 21, 22 Model assumptions on the efficacy [177Lu]Lu-DOTA-TATE in GI-NETs were based on the results of the phase III NETTER-1 trial , 14. ThiMedian PFS data for everolimus and sunitinib were based on the results of randomised phase III placebo-controlled trials in which patients received BSC in addition to assigned study treatment. The RADIANT-3 and RADIANT-4 trials compared everolimus with placebo in patients with PanNETs and GI-NETs, respectively , 10, whiThe main measure of safety used in the model was the incidence of grade 3 or 4 adverse events (AEs), applying the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) that were in use at the time each study was conducted. Data were abstracted from SmPCs , 21, 22;Costs were calculated from the perspective of the Italian national health service (NHS) over a time horizon of 1 year and are reported in 2020 euros (€) –26.For [177Lu]Lu-DOTA-TATE, acquisition costs were calculated for a complete course of therapy, defined according to the approved posology for the product and consisting of a total of up to four intravenous infusions of 7400 MBq each . The anaPatients with NETs routinely receive treatment with an SSA for the management of symptoms associated with functional tumours. Therefore, the acquisition costs of supportive treatment with SSAs were calculated for 1 year’s treatment for all three therapies. In the case of everolimus and sunitinib, costs were based on an ‘average’ dose of long-acting SSAs  and an assumed adherence rate of 100%. For [177Lu]Lu-DOTA-TATE, these costs were calculated as the sum of long-acting SSAs costs for 36 weeks and costs of short-acting SSAs for each 4-week period  prior to treatment, in line with the SmPC  and the Additionally, 177Lu]Lu-DOTA-TATE must be co-administered with an intravenous amino acid infusion to reduce the radiation dose absorbed by the kidneys and, consequently, the risk of renal toxicity  had extensive experience in the treatment of NETs considered reflective of current clinical practice in Italy. Hospital admission frequencies were based on clinical practice at these centres. Estimates used in the model were based on consensus among the clinical experts.The estimated frequencies of resources used for the management of adverse events  were subsequently converted into costs using the tariffs associated with the relevant ambulatory and hospital services.In particular, to calculate the costs associated with specialist appointments and/or hospitalisations resulting from grade 3 or 4 AEs, current tariffs for specialist ambulatory services and for DRG-related hospital services were used. For ambulatory service costs, the most recent versions of the National Tariff for Hospital Services for Acute Patients  and the The base-case assumption was that any grade 3 or 4 AE would prompt at least one consultation at a specialist centre. The cost of these visits was calculated as the mean of the tariffs in the National  and RegiThe purpose of sensitivity analysis is to investigate the degree to which the conclusions are sensitive to changes in the assumptions that underpin the model. However, sensitivity analyses tend to be arbitrary and may not reflect reality; for example, drug acquisition costs used in the base-case analysis are official prices minus mandatory discounts, but discounts in real-world practice may be greater. Thus, instead of presenting our own sensitivity analyses, conducted by adjusting key variables by predetermined amounts or percentages, we developed a simple, flexible tool that allows healthcare professionals and payers in Italy to input data relevant to their own institutions; this allows immediate generation of institution-specific cost-consequence data for each treatment option.What discount would be needed for everolimus  and sunitinib to match the modelled costs per progression-free month obtained in the base-case simulation for [177Lu]Lu-DOTA-TATE?How would the modelled costs per progression-free month change if the dose intensity or adherence assumed for the three treatments were lower than 100%?How would the modelled costs per progression-free month change if patients receiving [177Lu]Lu-DOTA-TATE were treated in a day-hospital setting, instead of being hospitalised for at least 1 day? In this case, a reduced tariff of €353 for DRG 409 was applied.Given the flexibility of the model, several simulations were performed to address specific questions of potential interest to real-world users in the context of the Italian NHS. Following discussion in an ad hoc working session, the authors identified the following questions, based on expected changes in drug acquisition costs, real-world experience with regard to variance in dose exposure and adherence, and potential changes in hospitalisation modalities for patients receiving [177Lu]Lu-DOTA-TATE:In each simulation, all other variables were kept constant, in order to explore the impact of each specific factor on the end result.For [177Lu]Lu-DOTA-TATE, the median PFS used in the model was 28.4 months in patients with GI-NETs and 30.3 months in patients with PanNETs . Median 3In total, 26 different grade 3 or 4 AEs were identified and mapped to DRG hospital codes Table . The resWith the exception of haematological AEs, the incidence of individual grade 3 or 4 AEs in the NETTER-1 trial was similar for the active and control groups . HaematoCosts associated with each treatment option, and modelled costs per progression-free month, calculated in the base-case scenario, are shown in Table In PanNETs, the purchase-price discounts required to match the base-case cost per progression-free month obtained for [177Lu]Lu-DOTA-TATE were calculated to be 52.7% for originator everolimus, 21.4% for generic everolimus, and 56.3% for sunitinib. For GI-NETs, the discounts required were 47.7% for originator everolimus and 13.0% for generic everolimus.We also explored the effects of variations in dose intensity and adherence. Assuming that dose intensity for [177Lu]Lu-DOTA-TATE was equal to the actual mean number of doses in NETTER-1 (i.e. 3.55 doses) and that adherence to everolimus and sunitinib was 85%, costs per progression-free month in the treatment of PanNETs were €2687 for [177Lu]Lu-DOTA-TATE, €4231 for originator everolimus, €2954 for generic everolimus, and €4538 for sunitinib. In GI-NETs, costs were €2867 for [177Lu]Lu-DOTA-TATE, €4244 for originator everolimus, and €2963 for generic everolimus.Adjusting dose intensity/adherence for all products to 75%, costs per progression-free month in the treatment of PanNETs were €2318 for [177Lu]Lu-DOTA-TATE, €3735 for originator everolimus, €2608 for generic everolimus, and €4006 for sunitinib for PanNETs. For GI-NETs, costs per progression-free month were €2473 for [177Lu]Lu-DOTA-TATE, €3746 for originator everolimus, and €2616 for generic everolimus.Lastly, changing the length of hospitalisation for [177Lu]Lu-DOTA-TATE administration to < 1 day reduced the cost of this treatment option (per month of PFS) to €2842 in PanNETs and to €3032 in GI-NETs.Overall, the results were most sensitive to variations in drug acquisition costs and adherence rates. Variations in the costs of managing AEs did not markedly influence the results (data not shown).Economic evaluation has become an important step in the integration of new technologies into medical practice and forms the basis of reimbursement decisions in countries  where healthcare resources are finite. Such evaluation usually measures the costs and consequences of the new technology against the existing standard of care and enumerates its incremental cost per unit of benefit gained. Essentially, the choice of a specific methodological approach depends on the kind of decision that the analysis is intended to inform: the greater the complexity of the decision , the more complex the methodological approach will be.In Italy, [177Lu]Lu-DOTA-TATE, everolimus, and sunitinib are recognised as innovative therapies and, accordingly, are reimbursed by the Italian NHS for GI-NETs and/or PanNETs –32; thisOur cost-consequence model was developed with these considerations in mind: the intervention of interest ([177Lu]Lu-DOTA-TATE) was compared with two alternative treatments in terms of costs per progression-free month; published median PFS data and grade 3/4 AEs were included in the model because these are the variables with the greatest influence on treatment selection in patients with GEP-NETs. Everolimus (indicated for both PanNETs and GI-NETs) and sunitinib (indicated for PanNETs only) were included in the analysis because they are the only approved alternatives to [177Lu]Lu-DOTA-TATE for progressive GEP-NETs  in Italy; high-dose octreotide LAR is not approved in these indications and therefore was not included in the analysis.We found that [177Lu]Lu-DOTA-TATE had higher acquisition and treatment costs than sunitinib or everolimus but resulted in markedly longer median PFS than either of the oral agents. In both the base-case and sensitivity analyses, this translated into lower costs per progression-free month versus comparators.We believe that our study is the first pharmacoeconomic comparison of systemic treatments for GEP-NETs in Italy. Economic analyses of [177Lu]Lu-DOTA-TATE have been reported in conjunction with reimbursement dossiers. In one analysis, conducted from the perspective of the French healthcare system, [177Lu]Lu-DOTA-TATE was associated with incremental costs of €42 106 per quality-adjusted life-year (QALY) gained versus high-dose octreotide LAR and €59 769 per QALY gained versus everolimus in the treatment of advanced GI-NETs . The autn = 443) had an overall median PFS of 29 months Lu-DOTA-TATE in the treatment of GEP-NETs is limited, although a study is planned in Italy. In the Erasmus phase I/II study, patients with bronchial NETs or GEP-NETs who received ≥ 22.2 GBq of [177Lu]Lu-DOTA-TATE  and/or sunitinib in both indications.We also investigated the level of purchase-price discount that would be required, in both PanNETs and GI-NETs, for everolimus and/or sunitinib to match the base-case cost of [177Lu]Lu-DOTA-TATE per progression-free month. In the treatment of PanNETs, discounts of > 50% were needed for originator everolimus and sunitinib, whereas reductions of > 20% were needed for generic everolimus. In GI-NETs, discounts of 48% for originator everolimus and 13% for generic everolimus would be required.The costs of [177Lu]Lu-DOTA-TATE per progression-free month were further reduced if treatment was administered in a day-hospital instead of an in-patient setting. Our model was specifically designed so that users could input costs for the duration of hospitalisation relevant to their institution or jurisdiction, reflecting either a conservative  or non-conservative  scenario.Another consideration is that the hospital costs associated with administration of [177Lu]Lu-DOTA-TATE in a day-hospital setting were quantified using the national tariff , resultiWe did not adjust our results for the effects of treatment on health-related quality of life (QoL). In the NETTER-1 trial, [177Lu]Lu-DOTA-TATE was associated with a statistically and clinically significant QoL benefit (i.e. a significantly longer time to deterioration of EORTC QLQ-C30 and G.I.NET-21 scores) versus high-dose octreotide . FurtherPatient preference is increasingly important in medical decision-making and is a complex construct resulting from the interplay between drug  and patient-specific factors . It is a separate area of study and, although highly relevant, has not yet been included in our model.90Y and/or 177Lu peptide receptor radionuclide therapy in a large series (n = 807) of patients with NETs treated at the European Institute of Oncology in Milan can be regarded as supportive [One possibly contentious assumption used in our model was that the costs of follow-up would be the same for [177Lu]Lu-DOTA-TATE as for everolimus and sunitinib. It could be argued that exposure to [177Lu]Lu-DOTA-TATE might pose an increased risk of long-term renal or haematological complications relative to everolimus or sunitinib, which may necessitate a closer follow-up of the patient. Follow-up assumptions made in the model were based on published evidence and consensus of Italian experts reflecting current clinical practice in Italy. Further, a long-term safety analysis of the NETTER-1 study revealed a benign safety profile with no new safety signals of any kind . The lonpportive . Thus, wpportive , characttout court to other settings without prior customisation. The estimated costs associated with the management of adverse events of the three therapeutic approaches under evaluation should be further validated through detailed research and analysis of real-world data from clinical practice in the Italian healthcare setting, such as NCT04727723  for [177Lu]Lu-DOTA-TATE. However, considering the relatively low frequency of grade 3–4 adverse events for all three therapies, we do not anticipate this to significantly affect the end results of the overall analysis.Lastly, the results of our analysis reflect the perspective of the Italian NHS, and the experience of only three centres  within it, and cannot necessarily be applied In conclusion, from the perspective of the Italian NHS, [177Lu]Lu-DOTA-TATE was associated with lower costs per progression-free month than either everolimus or sunitinib in the treatment of advanced GEP-NETs, by virtue of its markedly longer median PFS, according to the results of our cost-consequence model that included AE-related costs. This model may help to guide informed treatment choices in patients with advanced GEP-NETs based on clinical and economic parameters.Supplementary file1 (DOCX 44 KB)Below is the link to the electronic supplementary material.


Potato virus S (PVS), Potato virus A (PVA), and Potato virus M (PVM) infections from three potato cultivars. Chemotherapy treatments were undertaken on in vitro shoot segments for four weeks in culture medium supplemented with 100 mg L−1 ribavirin. Thermotherapy on in vitro shoot segments was applied for two weeks at 40°C (day) and 28°C (night) with a 16 h photoperiod. Plant vitrification solution 2 (PVS2) and cryotherapy treatments included a shoot tip preculture followed by exposure to PVS2 either without or with liquid nitrogen  treatment. The virus status of control and recovered plants following therapies was assessed in post-regeneration culture after 3 months and then retested in plants after they had been growing in a greenhouse for a further 3 months. Microtuber production was investigated using in vitro virus-free and virus-infected segments. We found that thermotherapy and cryotherapy (60 min PVS2 + LN) used alone were not effective in virus eradication, while chemotherapy was better but with variable efficacy (20–100%). The most effective result (70–100% virus eradication) was obtained by combining chemotherapy with cryotherapy, or by consecutive chemotherapy, combined chemotherapy and thermotherapy, then cryotherapy treatments irrespective of cultivar. Regrowth following the two best virus eradication treatments was similar ranging from 8.6 to 29% across the three cultivars. The importance of virus removal on yield was reflected in “Dunluce” free of PVS having higher numbers of microtubers and in “V500’ free of PVS and PVA having a greater proportion of microtubers > 5 mm. Our improved procedure has potential for producing virus-free planting material for the potato industry. It could also underpin the global exchange of virus-free germplasm for conservation and breeding programs.Certain viruses dramatically affect yield and quality of potatoes and have proved difficult to eradicate with current approaches. Here, we describe a reliable and efficient virus eradication method that is high throughput and more efficacious at producing virus-free potato plants than current reported methods. Thermotherapy, chemotherapy, and cryotherapy treatments were tested alone and in combination for ability to eradicate single and mixed  Solanum tuberosum) is the world’s third most important food crop after rice and wheat, and is the staple food of 1.3 billion people , Potato virus A , and Potato virus S  are typically the most significant viral pathogens (either as single or as mixed infections) associated with substantial production losses  and Potato virus Y (PVY) have been successfully eliminated with high efficacy using cryogenic treatments  cultivars “Dunluce,” “Tahi,” and “V500” were obtained from the Lincoln site of PFR and used in this study. Infected in vitro stock plants with PVM, PVA, and PVS were previously identified and confirmed using the reverse-transcription polymerase chain reaction (RT-qPCR) method (see below) prior to the experiments outlined here. Virus-infected cultures  were multiplied and maintained in an actively growing state on basal medium (BM) composed of half-strength −1 sucrose and 7.5 g L−1 agar. For all experiments, the pH of culture media was adjusted to 6 prior to autoclaving at 121°C for 20 min. Cultures were incubated in a growth room maintained at 24°C with a 16 h photoperiod at a photosynthetic flux density of 40 μmol s−1 m−2 provided by cool-white fluorescent tubes . Subculture into fresh BM was performed every 4 weeks throughout the experiment.Tissue-cultured plants of three potato  obtained from 3-week-old in vitro stock plants and placed on 90 × 15 mm disposable polystyrene Petri plates containing 25 ml of BM at a density of 30 to 40 nodal sections per plate. The nodal sections were cultured under standard conditions to provide uniform explant material for the therapies. The apical shoot tips (1 mm) for cryotherapy were excised from the nodal sections after 1 week, and the shoots (1.5 cm) for chemotherapy/thermotherapy experiments were excised from the nodal sections after 2 weeks. The explants used for −1 sucrose, 0.05 mg L−1 indole-3-acetic acid, 0.05 mg L−1 gibberellic acid, 0.3 mg L−1 zeatin, and 6 g L−1 agar at pH 5.7;  were excised from 1-week-old shoots and cultured on recovery medium (RM) consisting of MS macro and micro elements, LS vitamins, 25 g LUniform apical shoot tips (1 mm length) were excised from 1-week-old shoots and transferred to liquid preculture medium 1  and maintained for 24 h on a shaker (50 rpm) in darkness at 24°C. They were then transferred to liquid preculture medium 2  and maintained under the same conditions for another 16 h. Precultured shoot tips were treated with plant vitrification solution 2 (PVS2)   at 22°C −1 agar) overnight in the dark and then transferred to RM. Cultures were maintained 1 week in the dark at 24°C, before being transferred to standard conditions.Following PVS2 treatment, the shoot tips were washed with unloading solution  pre-warmed to 40°C and maintained in the solution for 20 min at room temperature (22°C). Thereafter, for recovery, shoot tips were transferred to an intermediate recovery medium  in “Tahi” and “V500” were assessed.For cryotherapy (Cryo), similar steps to those described in “PVS2” were followed, and 2 minutes before the end of PVS2 incubations, PVS2-treated shoot tips were placed into a thin layer of PVS2 on sterile aluminum foil strips (~ 6 × 25 mm) and then plunged into LN. After 1 hour of LN exposure, the aluminum foil strips with shoot tips were thawed quickly by inverting the strips into unloading solution and recovered as described in “PVS2” above.−1 m−2 provided by cool-white fluorescent tubes. The shoots were grown in an alternating temperature regime of 28°C for 8 h in darkness and 40°C for 16 h with light for 2 weeks. Apical shoot tips (1 mm length) were excised from heat-treated shoots and cultured on RM in the dark at 24°C for 1 week before transfer to standard conditions.Apical shoot segments (1–1.5 cm in length) were excised from 2-week-old shoots and placed in 98 × 60 mm disposable polystyrene clear tissue culture vessels with 50 ml BM at a density of 15 nodal sections per culture vessel. After 2 days of culture under standard conditions, the culture vessels containing shoot segments were moved into a growth chamber with 40% relative humidity at a photosynthetic photon flux density of 70 μmol sApical shoot segments (1–1.5 cm in length) were subjected to thermotherapy, as described in “Thermotherapy”, after which apical shoot tips (1 mm length) were excised and subjected to PVS2 treatment (PVS2) as described in “PVS2”.Apical shoot segments (1–1.5 cm in length) were subjected to thermotherapy, as described in “Thermotherapy”, after which apical shoot tips (1 mm length) were excised and subjected to cryotherapy (Cryo) as described in “Cryotherapy”.−1 ribavirin  at a density of 15 nodal sections per culture vessel. Ribavirin was filter-sterilized and added to the medium after autoclaving. The cultures were maintained under standard conditions. After 4 weeks of ribavirin treatment, apical shoot tips (1 mm length) were excised and placed on RM and incubated in the dark at 24°C for 1 week before transfer to standard conditions.Apical shoot segments (1–1.5 cm in length) were excised from 2-week-old shoots and placed in 98 × 60 mm disposable polystyrene clear tissue culture vessels containing 50 ml BM supplemented with 100 mg L−1 ribavirin for 4 weeks under standard conditions, as described in “Chemotherapy”, after which apical shoot tips (1 mm length) were excised and subjected to PVS2 treatment (PVS2) as described in “PVS2”.Apical shoot segments (1–1.5 cm in length) were cultured on BM supplemented with 100 mg L−1 ribavirin for 4 weeks under standard conditions, as described in “Chemotherapy”, after which apical shoot tips (1 mm length) were excised and subjected to cryotherapy (Cryo) as described in “Cryotherapy”.Apical shoot segments (1–1.5 cm in length) were cultured on BM supplemented with 100 mg L−1 ribavirin for 2 weeks under standard conditions, as described in “Chemotherapy”, after which the tissue culture vessel containing the shoots in ribavirin medium was transferred to thermotherapy conditions as described in “Thermotherapy” for additional 2 weeks, followed by apical shoot tip isolation (1 mm length) and recovery process as described in “Thermotherapy”.Apical shoot segments (1–1.5 cm in length) were cultured on BM supplemented with 100 mg LApical shoot segments (1–1.5 cm in length) were treated as described in “Chemotherapy Followed by Combined Chemotherapy and Thermotherapy [C + (C + T)]” above, after which apical shoot tips (1 mm length) were excised and subjected to PVS2 treatment (PVS2), as described in “PVS2”.Apical shoot segments (1–1.5 cm in length) were treated as described in “Chemotherapy Followed by Combined Chemotherapy and Thermotherapy [C + (C + T)]”, after which apical shoot tips (1 mm length) were excised and subjected to cryotherapy (Cryo), as described in “Cryotherapy”.Shoot tip survival  gene transcript (sec3) were used as the plant positive controls for cDNA synthesis. RT-qPCR was performed on nucleic acids isolated from young leaves of tissue-cultured plantlets and greenhouse-grown plants by the commercial company SlipStream Automationnad5 were performed in 5.5 μl volumes containing 0.9 x Roche Lightcycler 480 Master Mix,sec3 were performed in 7 μl reactions using 1 x Roche Lightcycler 480 Master Mix, 3.12 mM Mg2+, 400 nM primers, and 2 μl cDNA. Prior to amplification, the cDNA was denatured at 95°C for 10 min and then cycled 45 times with 95°C/15 s, 60°C/15 s, and 72°C/30s. Following PCR, melt curve analysis was performed at 95°C/60s and 40°C/60s, followed by heating to 65°C and then to 95°C with continuous data acquisition at 25 acquisitions per degree Celsius. Results were scored with LightCycler 480 software release 1.5.1.62 SP2 using the “Abs Quant/Fit Points” analysis with the Noise Band fixed at a fluorescence value of 5.The virus status of plant materials was tested using RT-qPCR. Primers and probes  were puranscript  and the in vitro therapies and controls were assessed at two different stages: firstly of cultures post-regeneration, 3 months after therapy treatments  on BM under standard conditions for 2 weeks to initiate rooting. The rooted plantlets were then transferred to individual 12-cm diameter pots (800 ml capacity) containing a commercial substrate Daltons® potting mix  in an insect-proof greenhouse at 22 ± 2°C with a 12–14 h photoperiod under natural light. Plants were individually covered with insect-proof net to prevent possible virus-cross contamination , containing two to three fully expanded mination . After 3In vitro microtubers were induced using the method of in vitro virus-free and virus-infected stock plants. Five shoot segments were placed into 100 ml liquid BM in a 500-mL Erlenmeyer flask and were cultured on a shaker (75 rpm) under standard conditions for 2 weeks (−1 sucrose and 4 mg L−1 kinetin. Data on the number of microtubers produced per vessel and the size of microtubers (diameter) were recorded after 2 weeks of culture in liquid MPM  with logit link (logistic regression). For each treatment, the results were presented as the number of samples, the percentage success, and a 95% confidence interval for the percentage. Confidence limits for the predicted treatment means were calculated from the logistic regression results except for those means of 100% for which the confidence limits were replaced by limits calculated using the Wilson Score method . Compariin vitro therapies and controls were randomly selected from the population and virus tested using RT-qPCR. The total number of plantlets used for virus testing varied depending on the number of samples obtained in each treatment.The numbers of microtubers produced per vessel from virus-free and virus-infected sources were compared using one-way ANOVA separately for each cultivar., and the proportions of microtubers in each size class  were analyzed using a binomial GLM with a logit link separately for each cultivar and size class . All anaAll the shoot tips in the controls survived and at least 96.8% regenerated shoots . All theThe effectiveness of virus eradication differed among the virus species: PVA was more readily eradicated than PVS and PVM, regardless of the method used . CryotheThermotherapy alone did not produce any virus-free plants in “Dunluce” and “Tahi”; meanwhile, in cultivar “V500,” one in 10 plants was free of PVS and PVM. Interestingly, two out of 10 thermotherapy-derived “V500” plants were free of PVM after 3 months in tissue culture but one subsequently tested positive for PVM after further plant growth in the greenhouse .in vitro culture post-regeneration. However, one of the progeny from this sample tested positive for PVM later in the greenhouse  across the three potato cultivars with shoot regrowth varying from 48.3 to 90%, the lowest of which was for “V500” and the highest for “Dunluce” . Chemotheenhouse .Improvements on virus eradication were achieved through consecutive chemotherapy followed by combined chemotherapy and thermotherapy [C + (C + T)] treatments, with 50% of the evaluated “Dunluce” plants being free of PVS, 60% of the “Tahi” plants being free of PVS and PVA in mixed infection, and 40% of the “V500” plants being free of PVS and PVM in mixed infection .Thermotherapy or chemotherapy treatments followed by cryotherapy and the combination of all treatments affected shoot tip survival and regrowth in all three potato cultivars assessed . When thThe efficiency of virus eradication using thermotherapy followed by cryotherapy differed between virus species and cultivar. While 70% of the “Dunluce” plants evaluated were PVS-free, only 29% of the “Tahi” plants evaluated were free from both PVS and PVA, and 20% of the “V500” plants were free of PVS and PVM .Shoots exposed to chemotherapy followed by combined chemotherapy plus thermotherapy; then, cryotherapy {[C + (C + T)] + Cryo} had significantly decreased regrowth compared to the control or to shoots received individual treatments . AlthougVirus-free plants, at low frequencies, could be found in shoots subjected to thermo- or/and chemo-therapy followed by PVS2 exposure for 60 min without freezing in LN (PVS2 treatment). However, the efficiency of virus eradication was much higher when shoot tips were thermo- or/and chemo-treated followed by LN exposure Cryo; .Survival and regrowth of shoot tips decreased as durations of PVS2 treatment increased, for all three cultivars. Extended PVS2 exposure duration of 135 min resulted in the least shoot tip survival and regrowth, even without LN exposure .Shoot tip survival of “Dunluce” was between 48 to 100% and 42 to 100% across the seven PVS2 exposure times for non-LN (without freezing in LN) treated and LN-treated (Cryo) explants, respectively . RegrowtThere were no significant differences in regrowth between shoot tips of “V500” and “Tahi” that were immersed in LN and those without LN immersion (PVS2 treatment) for the PVS2 exposure durations of 60 and 135 min . Shoot rin vitro and survived the acclimatization process. No visible morphological difference was apparent in the plants following 3 months’ growth in the greenhouse. Except for two “V500” plants, all the plants that were virus-free at the in vitro stage were found to have a virus-free status after 3 months in the greenhouse. The two “V500” plants (from mixed-infected with PVS and PVM) tested positive for PVM; one was derived from thermotherapy alone and the other from chemotherapy alone  than in PVS-free plantlets , or chemotherapy treatment prior to combining chemotherapy treatment with thermotherapy followed by cryotherapy {[C + (C + T)] + Cryo} resulted in high efficiency of PVS, PVA, and PVM eradication; 70–100% across the three potato cultivars tested. Though the regrowth percentage was low in shoots exposed to chemotherapy followed by cryotherapy (8.6–28.8%), high levels of virus-free plants were obtained. Indeed, plants of “Tahi” were 100% free from PVS and PVA, “Dunluce” were 90% PVS-free, and “V500” were 70% free from mixed infection of PVS and PVM. The combination of all treatments resulted in similar shoot regrowth (10.7–29%) across the three cultivars with virus eradication rates of between 70 and 100%: with rates for “Tahi” (mixed-infected with PVS and PVA) being lower than “V500” (mixed-infected with PVS and PVM) which was lower than “Dunluce” (PVS-infected). Although the efficacy of these two treatments in eradicating viruses was similar, incorporating thermotherapy was more time-consuming and required a temperature-controlled growth chamber; therefore, our study shows that the combination of chemotherapy and cryotherapy is a more appropriate strategy for eradication of PVS, PVM, and PVA from infected in vitro potato plants.In this study, we combined several virus eradication methods  to efficiently eradicate single and mixed PVS, PVA, and PVM infection from virus-infected in vitro potato shoots. In shoot tip culture, the size of excised shoot tip affects to the success of virus eradication (Potato virus X (PVX) have been eradicated by 0.2 mm shoot tip culture from in vitro diseased shoots  completely failed to eradicate single infections of PVS, mixed infections of PVS and PVA, and PVS and PVM from infected dication ; our resdication . Previoud shoots . Althougd shoots . High ted shoots . Wang etd shoots .−1 ribavirin for 4 weeks, followed by cryotherapy. Previously, in vitro shoots of potato. In their study, PVM and PVS were successfully eradicated from potato cultivars “Tamyr” and “Nartau” after three subcultures  on medium with 100 mg L−1 ribavirin followed by cryotherapy. In our study, in addition to PVM and PVS, we also successfully eradicated PVA by combining chemotherapy and cryotherapy. Since our procedure did not require such a long period of culture on ribavirin, it could provide a higher throughput and more cost effective technique, as well as reducing the potential for somaclonal variation through reducing the number of culture steps. In our study, 1-mm apical shoot tips were excised from plants treated with ribavirin for 4 weeks, then subjected to droplet-vitrification cryotherapy using 60 min PVS2 exposure at 22°C; in contrast, The combination of chemotherapy and cryotherapy reported here involved treatment of the shoots in medium containing 100 mg Lin vitro infected plants in multiple species of economic importance (Potato spindle tuber viroid (PSTVd)-free plants from in vitro infected potato “Zihuabai” shoots. In our study, although cryotherapy resulted in low efficiency (20%) of PVA eradication in “Tahi,” the cryo-recovered plants were still PVS-infected. Recent research has shown that not all viruses can be eradicated using shoot tip cryotherapy, particularly in those that are able to infect regions within the meristem  was eradicated from diseased shoot tips independent of PVS2 exposure duration (50–100 min). Although, there was no influence of PVS2 exposure duration on GLRaV-3 eradication, it was evident that the long exposure to PVS2 (100 min) significantly reduced the percentage of cells surviving in the apical dome and leaf primordia. GLRaV-3 is a phloem-limited virus and, therefore, it might explain why Even though cryotherapy techniques are based on cryopreservation, unlike cryopreservation, cryotherapy protocols should seek to eliminate the maximum portion of differentiated and infected cells, particularly in plant species infected with viruses that are difficult to eradicate such as PVS and PVM. This is a balancing act: a strategy to improve virus eradication could be to use either shorter or longer PVS2 exposure durations than the optimal, but this may result in fewer cells from which to recover plants after LN exposure . Workingin vitro cultures of “Dunluce,” “Tahi,” and “V500.” This finding is consistent with results reported by in vitro. Negative effects on tuber yield of single or mixed infections of PVX + PVS have also been reported in greenhouse- and field-grown potato plants  derived from thermotherapy and chemotherapy treatments in this research and previously in raspberry and apple plants infected with Raspberry bushy dwarf virus and Apple hammerhead viroid, subjected to chemotherapy treatment  in potato cultivar “Dunluce.” Availability of a simple and efficient cryopreservation protocol that results in high levels of viability (≥ 40% after LN exposure), such as the one presented here, may facilitate the technology transfer between laboratories and the implementation of cryopreserved base collections .JB LM, RP, CW, DH, AM, and JN were employed by The New Zealand Institute for Plant and Food Research Limited.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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