Effects of ayahuasca on mental health and quality of life in naïve users: A longitudinal and cross-sectional study combination

Ayahuasca is a hallucinogenic decoction used as a traditional medicine in several Amazonian regions. The ritualistic use of ayahuasca has spread throughout many countries, making it necessary to study its risks and benefits. Two sub-studies were designed for this investigation. In sub-study 1, a psychiatric interview and a battery of questionnaires were administered to subjects (n = 40) before their first ayahuasca use. Two follow-ups were conducted at 1 and 6 months. In sub-study 2, the same interview and battery of questionnaires were administered to long-term ayahuasca users (n = 23) and their scores were compared with those of the ayahuasca-naïve group. In the first assessment, nearly half (45%) of the naïve users were found to meet the diagnostic criteria for a psychiatric disorder. After the ayahuasca use, more than 80% of those subjects showed clinical improvements that persisted at 6 months. The questionnaires showed significant reductions in depression and psychopathology. Regarding sub-study 2, long-term users showed lower depression scores, and higher scores for self-transcendence and quality of life, as compared to their peers in sub-study 1. Further controlled and observational naturalistic studies assessing the eventual risks and potential benefits of ayahuasca are warranted.


Results
For sub-study 1, we recruited 28 women (70%) and 12 men aged between 20 and 65 years (X = 35). The majority (95%) of subjects were from Spain. Regarding education, the majority (54%) of the sample graduated from university. Most participants were atheists (84%). The majority of the sample (59.2%) wanted to take ayahuasca for therapeutic purposes, mentioning emotional or psychological issues. Other motivations included self-exploration (29.6%), curiosity (11.1%), to have a psychedelic experience (11.1%), to receive insights regarding professional development (7.4%), or to start on a spiritual path (3.7%). Regarding the types of ceremonies that participants attended, 40% were neoshamanic (led by westerners who traveled to a South American country to learn the methods of traditional medicine), 37.5% religious (mainly Santo Daime, but also other religious groups derived from classical ayahuasca religions), and 22.5% psychotherapeutic (a typical Western approach, in which a psychotherapeutic setting is used, without religious or shamanic content) ceremonies. As is common in longitudinal studies, a smaller number of subjects was retained during follow-ups. While 28 subjects participated in the 1-month follow-up, 15 subjects participated in the 6-months follow-up. At baseline, eighteen subjects (45%; 77.7% women) met the criteria for one or more psychiatric disorders (50% with comorbidity). The most prevalent diagnoses were generalized anxiety disorder (GAD; n = 6) and substance abuse/dependence (alcohol = 2; cannabis = 2; lorazepam = 1). See Table 1.
At the one-month follow-up, the 18 subjects (77.7% women) who met diagnostic criteria were interviewed again. Eleven of them (61%) no longer met the criteria for any psychiatric disorder (0% of comorbidity). Four of these subjects (22.2%) presented fewer psychiatric diagnoses than at baseline. One subject (5.5%) met the criteria for a different psychiatric diagnosis than at baseline. Finally, one subject (5.5%) who did not meet the criteria for any psychiatric disorder at baseline did so during the follow-up, meeting the criteria for GAD. Thus, 7 subjects met the criteria for psychiatric diagnosis at this time-point. One subject had taken ayahuasca once more since the first assessment.
At 6-months follow-up, 15 subjects (60% women) continued their participation in the study. Five subjects took ayahuasca again since the 1-month follow-up. Only 8 subjects from the initial 18 who met the criteria for a psychiatric disorder were interviewed. Among those 18 subjects, only 2 still met the criteria for psychiatric diagnosis. According to the available information, the subjects who met diagnostic criteria at baseline but did not at the 1-month follow-up continued to not present diagnostic criteria for any psychiatric disorder. The participant who did not meet the diagnostic criteria for a psychiatric disorder at baseline but met the criteria for GAD at 1-month follow-up still met the diagnostic criteria for GAD at 6-months follow-up. No other subjects met the criteria for a psychiatric disorder, as only 3 subjects met the criteria at the 6-months follow-up.
Regarding the data from the administered questionnaires, at one-month follow-up, lower scores were found for two scales of the Symptom Check-List-90-Revised (SCL-90-R): anxiety (d = 0.54), and hostility (d = 0.62). At 6-months follow-up, lower scores were obtained for the Hamilton Depression Rating Scale (HAM-D) (d = 0.72) and in the role-emotional scale (d = 0.74) from the SF-36 questionnaire. See Table 2.
Regarding the subgroup analysis of data from the sample that met criteria for diagnosing psychiatric disorders, all of the CIs overlapped between assessments. However, a tendency was observed in scores obtained by most of the questionnaires, as they decreased significantly (data not shown).
Regarding the analysis of potential differences between subjects who dropped out of the study and those who remained, there were no significant differences in any variables, neither at one-month nor at the 6-months follow-up. However, some tendencies with notable effect sizes were registered. At one-month follow-up, subjects who dropped out of the study tended to obtain lower scores in CAPE-depression symptoms frequency Sub-study 2 was conducted in order to determine if any differences in assessed variables were due to ayahuasca use or previous conditions. In this sub-study, the baseline data of the sample for sub-study 1 were compared with long-term ayahuasca users' results for the same variables. We recruited 10 women (43%) and 13 men aged between 32 and 64 years (X = 45). The majority (78.3%) of the subjects were from Spain. Regarding education, the majority (32%) of the sample graduated from university. In terms of religious beliefs, most participants were atheists (34.8%). The mean number of ayahuasca ceremonies that participants attended was 70 (ranging from 50 to 100 ceremonies). Only two participants (9%) met the criteria for a psychiatric disorder. One subject met the criteria for suicide risk (for attempted suicide), GAD, and obsessive-compulsive disorder (OCD); the other subject met the criteria for suicide risk (for past attempted suicide) and antisocial personality disorder. See Table 1.
Analyzing data collected through the questionnaires, we found statistically significant differences between long-term and ayahuasca naïve users in the HAM-D score [t(53.  Table 3. Regarding the subgroup analysis involving only the sub-study 1 participants who met the criteria for a psychiatric disorder, more differences were found between those participants and the sample of long-term ayahuasca users. Differences were found in HAM-D score [t (39) Table 3.

Discussion
Since ayahuasca use is expanding both internationally and locally in the Spanish context, the aim of this study was to observe the effects of ayahuasca on psychological and health variables in naïve ayahuasca users. This will help medical practitioners to understand possible adverse effects as well as potential therapeutic uses. www.nature.com/scientificreports www.nature.com/scientificreports/ We performed two sub-studies. Regarding sub-study 1, 40 ayahuasca-naïve subjects were assessed before using ayahuasca for the first time, and they were followed up with at 1 and 6 months after. Following the usual pattern of participants in complementary and alternative medicines 30 , the majority of the sample consisted of women with higher education.
The Mini-International Neuropsychiatric Interview (MINI) interview showed that 45% of the sample met the diagnostic criteria for a psychiatric disorder. This finding is in line with the main reasons reported by participants for attending ayahuasca ceremonies, which include the treatment of mental health problems and to achieve psychological well-being 27,31,32 . Remarkably, at the 1-month follow-up, 61% of participants who initially met the    www.nature.com/scientificreports www.nature.com/scientificreports/ diagnostic criteria no longer met the criteria for any psychiatric disorder. Additionally, 22.2% of participants showed a decrease in the number of psychiatric disorders for which they met the diagnostic criteria, reducing the high prevalence of comorbidity at baseline (50%) to 0% at the 1-month follow-up. Overall, 83.2% of participants reported a clinical improvement. This improvement lasted until the 6-months follow-up. This finding is surprising, considering that this clinical improvement reported in the psychiatric interviews was not fully identified by the questionnaires used. A reduction in psychiatric diagnoses based on clinical interviews after the initiation of ayahuasca use is consistent across studies 6,33 . These results are also consistent with a recent study published by our group where it was found that half of a large sample (n = 380) of long-term ayahuasca users reduced or eliminated their prescription drugs after they began to regularly use ayahuasca 9 .
Only one subject met some criteria for a new psychiatric diagnostic, specifically for GAD, after using ayahuasca for the first time. That subject met the criteria for this disorder at 6-months follow-up. There are several reports regarding adverse effects of ayahuasca, leading some of them to psychiatric diagnoses 29 . In order to obtain more information, the questionnaire scores for this case were checked, and it was noted that the scores on every psychopathology scale from the SCL-90-R decreased notably at 1-month follow-up. At 6-months follow-up, half of the scales of the SCL-90-R increased again, following the general trend of the whole sample. In the clinical interview conducted using the MINI, this subject mentioned that the ayahuasca experience went well, and the traumatic termination of a partnership that they were experiencing was a more probable cause of the anxious state.
Regarding changes in psychological and psychopathological variables, there were improvements in the HAM-D at the 6-months assessment, in anxiety and hostility from the SCL-90-R at the 1-month assessment, and in the role-emotional scale from the SF-36 (see Fig. 2). The only variable that showed consistent change in the 6-month study period was depression as measured by HAM-D, which improved at every assessment. Although differences between baseline and the 1-month follow-up did not reach statistical significance, the scores decreased by half. This improvement was more evident at the 6-months follow-up, where differences between measures reached statistical significance. This finding is in line with previous research in which ayahuasca showed antidepressant effects at 7 and 21 days after its controlled administration in a clinical setting [14][15][16] . Our sample was not clinical, but 45% of subjects met the criteria for a psychiatric disorder. The same pattern of improvement was observed in the role-emotional scale from the SF-36. A similar study with a sample similar to ours also found long-term improvements in the role-emotional and social function scales from the SF-36 questionnaire 25 .
Psychopathology scales of anxiety and hostility from the SCL-90-R only improved at 1-month, but not at the 6-months assessment. This tendency was observed for many scales, despite not being statistically significant. A general trend of improvement at 1-month follow-up was observed, while this improvement progressively decreased up until the 6-months follow-up. This can be clearly seen in Table 3. This pattern suggests that the potential therapeutic benefits of ayahuasca are temporary and do not persist in time, excepting for the case of depression. Several studies comparing ritual long-term ayahuasca users with non-users or with normative data have found better SCL-90-R scores and better scores for other psychopathological and psychological measures in users 12,13,33,34 . This may suggest that the long-term benefits of ayahuasca use depend on maintaining regular use.
The size of the sample decreased at both follow-ups. Therefore, we wanted to understand any potential limitations in that regard, so we conducted comparisons between drop-out subjects and those who remained in the study. Despite not obtaining significant results, subjects who scored lower on depression symptoms frequency (CAPE), and depression and hostility (SCL-90-R) at baseline tended to remain as study participants at least until the first follow-up. There were no significant differences nor notable tendencies between subjects who quit and those who remained until the last follow-up regarding any variable. This finding suggests that people with a high degree of distress may feel better after ayahuasca use and, subsequently, be more motivated to collaborate with researchers.
When only participants who met diagnostic criteria for a psychiatric disorder were analyzed, all the confidence intervals (CI) of variables overlapped between assessments. However, they indicated remarkable decrease over time in HAM-D scores, in the psychopathology scores measured by SCL-90-R at the 1-month follow-up, decrease in AAQ-II scores at each assessment, and increase of the WHOQOL-Bref score in the same manner. This also suggests an improvement in psychopathological status as it was also found using the MINI, but the sample may have been too small to observe significant differences using CIs. Despite reporting a clear tendency on the questionnaires used, this was not enough so that the CIs did not overlap.
In order to better understand if the eventual differences found between assessments in sub-study 1 were attributable to the use of ayahuasca or to extrapharmacological variables, including the passing of time, or if there are any pre-conditions that may lead a person to get involved in ayahuasca use, we performed sub-study 2. Only the measure of depression, assessed with the HAM-D, showed significant differences between non-users and long-term users, where the latter scored better than the former, a finding consistent with the long-term antidepressant effects of ayahuasca [14][15][16] . In previous studies, the acute administration of ayahuasca reduced the functional connectivity (FC) of the default mode network (DMN) 21 , which could be related to the improvements made on a depressed condition 35 . Moreover, it has been observed that the acute administration of the psychedelic tryptamine psilocybin also reduced the FC in the DMN in patients with MDD 36 and resulted in a decrease in depression scores that lasted for at least 6 months 37 . Therefore, both the lower scores found at months 1 and 6 in naïve users and the lower scores for depression found in our long-term users, as compared with the baseline scores of subjects from sub-study 1, may reflect an actual mid-term antidepressant effect of ayahuasca. Due to the complex effects of ayahuasca, both pharmacological and psychological, employing an integrated approach involving different disciplines would be necessary to explain its efficacy. It has been observed, for example, that ayahuasca acutely enhances mindfulness-related capacities, such as decentering or acceptance 38,39 , and psychological process variables such as personal values could also be involved in therapeutic outcomes 34 . These capacities seem to be maintained during the after-glow period and also have persistent effects over the long-term 40 . (2020) 10:4075 | https://doi.org/10.1038/s41598-020-61169-x www.nature.com/scientificreports www.nature.com/scientificreports/ Long-term effects may also be associated with the integration of the experience into normal life (e.g. by reconciling unexpressed/unresolved emotional energy), and thus with improvements in psychotherapeutic outcomes 41 . Furthermore, some authors suggest that the psychedelic experience 42 or the mystical-type experiences 43 that psychedelic drugs induce can also explain their therapeutic effects. Finally, as ayahuasca is generally taken in   Table 3. Means and standard deviations from the sub-study 2. The middle column shows the differences observed between ayahuasca-naïve subjects and long-term ayahuasca users. The column on the right shows the differences found between long-term users and the ayahuasca-naïve participants who met the criteria for diagnosing psychiatric disorders. www.nature.com/scientificreports www.nature.com/scientificreports/ ceremonies for which small or large groups of people gather 44 , its communal use can also be viewed as exerting potential benefits, since feeling part of a community can have an important therapeutic impact 10 .
The other measure where experienced users scored higher than naïve users was self-transcendence (ST). There are multiple studies that have shown that psychedelics can induce long-term personality changes 45 . In sub-study 1, subjects did not change their scores in ST between assessments, but in sub-study 2 long-term users scored significantly higher than non-users in ST, a result that has been consistently found in previous studies as well 11,12,24 . It has been suggested that ayahuasca users' higher ST scores could be partially explained by their participation in religious practices such as Santo Daime. However, in the study by Bouso et al. 12 the samples (ayahuasca users and non-users) were matched for religion, age, and gender, so the difference in that study could be attributed to the ritualistic use of ayahuasca. In the present study, the number of atheists was lower in the group of long-term ayahuasca users, but only 13% of participants were Santo Daime members. Taking all these data into account, it is reasonable to think that higher scores in ST may be a direct consequence of ayahuasca use. A previous study found differences in cortical thickness in several brain areas to exist between long-term users and non-users of ayahuasca, suggesting a physical brain modification after regular ayahuasca exposure without any apparent neuropsychiatric consequences (there were no differences between groups in their performance on psychiatric and neuropsychological tasks) 13 . In that study, researchers found a correlation between ST and cortical thickness in the posterior cingulate cortex 13 . Psychedelic drugs may therefore offer an interesting tool for investigating the stability of personality traits and their brain correlates.
Regarding the differences between naïve ayahuasca users' and experienced users' baseline scores for quality of life as assessed by the WHOQOL-Bref, they are also consistent between sub-studies. Although in sub-study 1 there were no significant differences between assessments, there was an actual clinical improvement between them (see Table 3). This is consistent with previous research where regular ayahuasca use was associated with a better mental health status or with improvement on psychopathology scales, as was also observed in sub-study 1.
When comparing long-term ayahuasca users with the sub-study 1 subsample of psychiatric diagnosis (n = 18), the number of variables in which significant differences are found increases. This is reasonable in light of the much smaller number of long-term ayahuasca users who met the diagnostic criteria for at least one psychiatric disorder (9%). Ayahuasca-naïve users who met the criteria for a psychiatric diagnosis scored poorly for depression (HAM-D), positive symptoms frequency (CAPE), depression symptoms distress (CAPE), negative symptoms distress, in AAQ-II questionnaire, self-transcendence scale (TCI-R-67), role-emotional (SF-36), and www.nature.com/scientificreports www.nature.com/scientificreports/ WHOQOL-Bref. Despite not finding statistically significant differences between assessments among the subgroup of naïve users with psychiatric disorders, remarkable improvements were observed for several scales measuring psychopathology and quality of life. This lends support to previous research that found that ayahuasca users gained therapeutic benefits or experienced an enhanced ability to solve personal problems 25,33,46 .
The main limitation of this study is that there was no control group with which to make comparisons to determine the impact of the intervention. In order to minimize that limitation, we designed sub-study 2. Coherent results were found both between sub-studies and with previously published research. However, further studies using larger samples should consider potential differences between the type and setting of the ceremony.
This study assessed naïve users of ayahuasca in order to collect data regarding its overall effects on psychiatric condition and psychological status. Since ayahuasca use has become globally popular as a self-help practice, it is important to better understand its potential risks and benefits. General mid-term adverse effects were not observed in this study, although some secondary acute reactions were observed in some individual cases (e.g. anxiety) and will be reported in a separate paper. Additionally, from a clinical point of view, there was a substantial decrease in psychiatric symptomatology after the first use of ayahuasca, which persisted until the 6-months follow-up. The most evident improvements were found with regards to depression. These improvements in depression found after performing clinical interviews were also demonstrated by the psychiatric rating scales. Better scores for depression were also observed among long-term users when compared with ayahuasca-naïve users at baseline. The results obtained in this study help to inform us about the effects of the ritualistic use of ayahuasca on mental health. These results suggest that the use of ayahuasca in controlled settings may offer therapeutic benefits. Future studies with larger samples are warranted in order to better understand 1) the more specific effects of ayahuasca use on public health and 2) the reasons why those initiated in ayahuasca use continue to use it or not.

Methods
Sub-study 1. Sample. Several ayahuasca ceremony leaders from different parts of Spain were contacted and asked to inform us when ayahuasca-naïve subjects called them to participate in their ayahuasca ceremonies. Subjects willing to participate contacted the authors. In this way we recruited 40 ayahuasca-naïve subjects (28 women) for sub-study 1.
Measures. Sociodemographic information. Age (years), gender (male/female), nationality, maximum educational level, religion, and motivation for participating in ayahuasca ceremonies were collected from participants.
Mini-International Neuropsychiatric Interview (MINI; Sheehan et al. 47 ; Spanish version: Ferrando et al. 48 ). MINI is a short structured diagnostic interview that includes DSM-IV and ICD-10 psychiatric disorders. It is a short but accurate tool for use in both clinical trials and epidemiological studies.
Hamilton Depression Rating Scale (HAM-D; Hamilton 49 ; Spanish version: Ramos-Brieva 50 ). HAM-D is a questionnaire used to rate the severity of depression through the assessment of various aspects, such as mood, suicide ideation, insomnia, anxiety, or somatic symptoms. A score of 0 to 7 is considered to be normal, while a score of >7 is indicative of depression.
Community Assessment of Psychic Experience (CAPE; Stefanis et al. 51 ; Spanish version: Fonseca-Pedrero et al. 52 ). CAPE was designed specifically to evaluate psychotic-like experiences in epidemiological studies in the general population. It contains 42 items and measures psychotic-like experiences using eight sub-scales (Total Frequency score; Total Distress score; Positive Symptoms Frequency; Positive Symptoms Distress; Depression Symptoms Frequency; Depression Symptoms Distress; Negative Symptoms Frequency; Negative Symptoms Distress) based on a dimensional approach.
Acceptance and Action Questionnaire (AAQ-II; Bond et al. 55 ; Spanish version: Ruiz et al. 56 ). AAQ-II is a measure of psychological flexibility that shows good test-retest reliabilities. High scores imply worse psychological flexibility.
The World Health Organization Quality of Life (WHOQOL-Bref; WHO 61 ; Spanish version: Lucas-Carrasco 62 ). WHOQOL-Bref is an abbreviated 26-item version of the WHOQOL-100. It is an assessment tool with cross-cultural validity that is used to assess the quality of life.