From the remaining 366, 277 (75.6%) responses were obtained, and 30 provided formal follow up information on more than 1 occasion. The group without baseline information contained more young people who were still at secondary school, thus reflecting the difference in age distribution and use of services (t-test for independent samples t = −3.33 df 508 p < 0.001 F-ratio variances 1.68 p < 0.00005). There was also a significant difference in mean duration of follow up 7.6 years sd 4.5 (range 1–21.6) years compared with 8.3 (1–19) years (p

GHQ 277 16.0 3.01 171 16.6 7.0 66 10.1 5.3 7.8 <0.0005

TABLE 1 | Comparison scores for Depression (Beck), Anxiety (Speilberger), and General Health Questionnaire (GHQ) for study group compared with previous clinical and community age and gender matched samples.

*aRowe and Rowe (22).*

< 0.05) and the proportion reporting recovery was significantly different 40% compared with 52% (Chi-square 6.8, p < 0.01).

As the majority of comparisons including: duration of illness (4.9 cf 5.1 years), functional score if reporting recovery (9.0 cf 8.8), duration of illness until help received (13.4 cf 13.8 months), proportion working or studying full time (61% cf 63%), proportion caring for children (5%) or not working or studying (5%) were not significantly different, the general comments and observations have been combined from the two groups (see **Table 2**).

#### Educational Outcomes

Only 5% of those followed up reported not working or studying, with 8% working or studying less than half time, 24% more than half time and 63% reported working or studying full time. Some of those reportedly not working or studying were traveling overseas prior to university whereas others remained very unwell or had other illnesses. Similarly, 62% reported that they were currently studying at either a secondary or tertiary level. Of the 66% who had undertaken a post-secondary course (tertiary education) half were still studying. Twenty per cent were also working part time while they were studying. Twenty per cent reported using the Visiting Teacher Service during their secondary education and 16.5% used Distance Education service either solely or supplementing their school attendance. Five per cent reported having children, some of whom were working outside of home as well.

For comparison, the 2011 census (18), indicated that 75% of 15–25 year olds complete secondary education and 60% undertake post-secondary education, with 6% unemployed, 9% not in the workforce and 26% in employment. The population census reported 85% of 15–25 year olds are fully engaged in either work or study or both.

A wide range of courses were undertaken by the cohort ranging from traditional University courses to ones with high entry criteria such as medicine, law and health sciences. Technical

#### TABLE 2 | Comparison data between study and comparison groups.

Total cohort *n* = 784 (M:F 1:3) Mean age 22.5 years (range 7–35.7 years) Follow up (FU) data from 641 (81.8%) Mean length FU 8 (1–21) years Proportion reporting recovery 46.5% Mean duration illness 5 years (1–15) (*n* = 298)

\* *t-test p* < *0.05* \*\**t-test p* < *0.001. #chi square 6.8 p* < *0.01. Bolding indicates composition and outcomes for total cohort.*

courses, diploma courses and some apprenticeships were also reported. Several had also completed Masters degrees and Ph.Ds. A very wide variety of occupations were reported from aeronautical engineering, health sciences, law, trades and with some working in the service industries. The majority of service industry jobs were part time for support while undertaking study. A very small percentage did not complete secondary education and this was reported associated with other illness, social circumstances or an unsupportive school.

Disability support from the government was received by 29% of the group. The majority was studying while receiving this or working part time. At the time, government policy allowed this support to enable part-time completion of a degree for those who were chronically unwell, without having to work as well. Of the 5% not studying or working outside of home, several were mothers, one was a carer for a parent, and others had an additional illness or were doing voluntary work.

### ME/CFS Functional Score and Reported Recovery

As some young people found the concept of recovery difficult, they were asked to describe how they thought they were functioning with a rating of 10 being "very well" or "back to normal" and 1 being "bedridden." They were also asked what they meant by their score and it usually included a sense of the amount of activity, work or study that they were able to manage, what their stamina was like, how well they recovered from any activity, as well as social engagement and presence of symptoms. The young people were remarkably consistent in what they included in the scoring and how they described it. The clinician also rated the young person based on their reports and found high inter rater reliability for the scores (90%). Parents were generally also asked and they agreed in 80% cases. They often rated one point below the young person as they tended to compare them with how they "used to be." The descriptors of the global rating are in **Appendix 3**. Young people reported that the central part of the Bell CFIDS Scale was difficult to score and they preferred to use the global rating (ME/CFS Functional rating). Concurrent data were collected to allow comparison and this difference in scoring in the mid range (between 4 and 8) was confirmed. The correlation was 0.833 (n = 252), however the correlation between the two scales was less in the mid-range (0.65) with the ME/CFS Functional Rating tending to score higher. The Bell scale had an irregular distribution with scores 9 and 10 scoring with higher frequency but low frequencies in the mid-range and an increase at the lower end.

The study group was asked to assess their poorest level of functioning using the Bell scale and 98.7% scored <5 while 68% scored 2 or less. They were also asked their current level of functioning using the same scale as well as the functional rating.

The range of ME/CFS Functional score for those who reported that they had recovered was compared with those who reported they had not recovered. (**Figures 5** and **6**) There was a significant overlap. Some scored low due to other illnesses that they differentiated from CFS. Others reported that they did not know what was "normal" as it had been so long since they were well. They reported that "how a 22 year old spent their energy was different from 15 year, but they were not sure what that should be." Others, who reported themselves as "well," felt that they were managing well, but their parents made the comment that they "did not think that they had the stamina that they had demonstrated as an adolescent." Some needed to be "perfect" to describe themselves as "well" whereas others compared themselves with how they were when they were first unwell and were very grateful to be able to do what they were currently able to do. Some who felt they had recovered also scored lower, as they were caring for small children, were working part time and felt sleep deprived. Others admitted that they were dealing with depression as an additional issue. Hence many factors influenced whether recovery was reported, as well as whether the level of functioning was solely related to ME/CFS.

### Reports of Recurrent Infections and Prolonged Recovery Afterwards

The majority reported a reduction in the number of infections and the fact that they rarely had upper respiratory infections when the rest of the family was unwell. They did notice that when they were starting to feel better they commonly had an increased number of upper respiratory infections over a year or two which then settled to a normal rate. This did trigger significant panic and low mood as the symptoms they experienced with these common illnesses reminded them of the early days of ME/CFS

and they felt that they could not deal with prolonged illness again. As these illnesses commonly recurred on return to school they acknowledged that it made them more cautious about engaging again. They also clearly did not believe reassurance that feedback from others who had experienced similar events, indicated that recovery is generally short lived. Comments on feedback forms indicated the fear and anxiety was excessive but not warranted. Those who had had regular asthma prior to becoming ill, observed that they rarely had asthma when they were very unwell with ME/CFS. Approximately 5% had documented EBV infection when still unwell but only one young person who had significantly improved at that stage, had a prolonged recovery of several years. The remainder reverted to their usually level of functioning in <3 months.

### Other Illnesses

The majority of associated illnesses reported were depression (rarely associated with depression at baseline), severe anxiety, panic disorder, and POTS and neurally mediated hypotension. There were individuals with agoraphobia, eating disorder, irritable bowel disease, celiac disease, Graves disease, Rheumatoid arthritis, and several developed Systemic Lupus Erythematosis (SLE) but all but one of these had negative ANA at the onset of CFS. Two struggled with drug addiction. One young woman had died at 22 from neuroendocrine carcinoma of the cervix and another had breast cancer at 23 who incidentally reported having a reduction in her CFS symptoms after chemotherapy. At least 10 young women disclosed sexual abuse years later, the majority of which occurred after they were unwell, thus adding to the trauma.

#### Members of Family With History of ME/ CFS

Although data from the baseline questionnaires indicated 17% had a close family member who had a prolonged recovery from an infection lasting more than 6 months, a diagnosis of CFS in the past or currently, only 12% of that group reported that a family member has had, or has ME/CFS. In contrast, 15% of the remaining cohort reported a family member with a history of ME/CFS. Parents, siblings and cousins were identified, with some families having three immediate family members with the diagnosis.

### Any Predictors of Outcomes? Presence of Positive Antinuclear Antibody Titers

The proportion of positive titers (>1:160) was at least double the expected rate in this age group (37, 38), and it is expected that titers increase with age. However, subsequently only 4 (0.5%) patients had diagnosed SLE. Of these, 2 had titers of 1:160 (speckled pattern) and one had a titer of 1:1,280 homogeneous pattern at age 15, but no other features. One with later rheumatoid arthritis and 2 with Sjogren's disease had negative ANA titers, while one who developed Grave's disease had a titer of 1:320 with speckled pattern.

There was no significant difference in reported recovery rate and level of ANA titer [General Linear model F(1, 76) = 0.12 p = 0.73]. Those with high titers (1,280–2,560) reported recovery from CFS. Of those with a titer of 1:640 or higher, there were no follow up data available for 25% of them. Of the remainder, in ¼ the titer remained stable, ½ the titer reduced to normal or near normal, and ¼ the titer increased without any other clinical features of disease. Therefore, high titers were not associated with "non-recovery" and there was a suggestion that titers reduced with recovery.

### Outcomes for Severe Symptoms at Outset

The rating for level of ill health early in the illness was a mean score of 1.9, sd 1.3 (range 0–5) with 98.7% scoring <5 and 68% 2 or less, using the Bell CFIDS score. At worst, the majority were bed ridden and unable to attend school at all, or at the most, attended for short periods during the week. Therefore, in this study the majority of young people had severe illness. Severity of illness was not predictive of "non-recovery."

### Relationship of Identified Depression, Anxiety, or GHQ Scores to Outcomes

There was no difference in depression score at baseline between those that reported improvement (mean 14.46) and those that had not (mean 13.17) df 315. t = −1.3 p = 0.18. Although data were available on follow up from 86% of those with Beck data there was no significant difference in baseline score between the total group and those followed up (mean1 = 13.85, mean2 = 13.78; df 688 p = 0.91). If selected for higher depression scores at baseline (20% of sample) there was no difference in baseline score between those that reported recovery and those that did not (means 28 and 27.5) i.e., depression at baseline was not predictive of outcome.

There is also no difference in anxiety score at baseline between those who reported recovery and those who did not, so anxiety at baseline was not predictive of outcome using student t-test (mean1 = 28.1, mean2 = 27.5; t = 0.4 p = 0.7).

Similarly, the GHQ score at baseline was not predictive of recovery (t-test −0.62, df 233, p = 0.5).

### Predictors of Good Functional Outcome

There were no obvious predictors for outcome. However, as the young people reported how important remaining engaged in education and socially connected, as well as feeling supported, encouraged and being believed, and how difficult it could be to deal with social isolation, social confidence and unsatisfying work when still unwell, it could be inferred that improving these circumstances improves their ability to cope with their situation and function better. Use of disability support during post-secondary education was very important, if they were not able to work as well as study part time. Ensuring that they could continue study at a reduced rate without having to try and find work to support themselves was key. Frequently when they had completed their studies and able to find a job, even if part time, they were often in a much better position to support themselves and feel independent.

### Feedback Regarding Management Feedback Regarding Helpful Professionals

Twenty three per cent of the group providing baseline information "did not find any professionals helpful," however only 17% of the remainder did not find professionals helpful despite our concerns that this group may contain a higher proportion of young people who did not engage with health services. Pediatrician, Visiting Teachers, cardiologists who were consulted because of orthostatic intolerance were most frequently cited, and less frequently physiotherapist, family doctors, some school staff, counselors, psychologist, massage therapist, sleep physician and gastroenterologist. The reasons they were found helpful related to "being taken seriously," "being believed," "providing support," "providing practical strategies," "alleviating symptoms," "providing educational liaison and advocacy," "dietary advice," and "managing other illnesses" that may also be present. The most common responses were appreciating "being understood," "feeling respected," "supported," "reassured," and "not feeling alone."

### Feedback Regarding Helpful Information

The young people consistently indicated that the most helpful information was providing some management strategies to help them "feel in control of their life again." Managing symptoms and providing information regarding the importance of good routines particularly around sleeping and eating and activity was also appreciated, as well as understanding how to monitor their activities. Strategies for re-engaging with peers, assistance with liaising with schools as well as providing information regarding options to remain engaged in education were rated highly. General information about the illness prognosis and what had helped others, as well as up to date research information was useful. In addition to the hospital clinic, information was sourced from the internet, ME/CFS Society newsletters, and the support groups.

### Could the Illness Have Been Handled Better?

There were some differences in feedback from the early questionnaires compared with the later ones mostly related to lack of awareness of the illness. They indicated the need for an earlier diagnosis, and more understanding by the community, the medical profession and by schools. They reported how important it is "to be believed" and to find "someone who understands." There were regular comments about the perceived arrogance of the medical profession and lack of understanding, and how distressing it was to be accused of "being lazy" or implying that it was "all in the mind." Lack of understanding and flexibility by educational institutions was reported as a major source of distress.

In later follow up returns, the comments regarding the need for general understanding of the illness and their plight, the need for earlier diagnosis and access to management strategies were similar. There were comments regarding self-management that they thought they could have managed better, such as managing stress, routines, pacing, depression, exercise and being more open to acknowledging the presence of depression especially as they were improving and re-engaging in society. There were comments regarding the need for resources to be available for the family, as well as regular but not necessarily frequent follow up appointments, and that contact with other young people in a similar situation would be helpful. Many commented that they also needed to be willing to ask for help.

Many indicated the need to be sensitive about when psychological assistance is offered. They were often not ready for that intervention as they felt they had enough to cope with. They were sensitive as to whether this was implying that psychological issues were the "cause." Many indicated that they were "miserable" and "fed-up with being unwell" but not depressed which on reflection was a healthy response to their situation. If they were happy and content with not being able to attend school, see friends, or do any physical activity it would be a cause for concern. Others reported that they struggled with motivation and seeing any end to the illness. Some had a family history of depression and were open to receiving assistance. It was noted that when many young people started to feel a little better they were willing to have strategies for managing stress and anxiety and also reassurance that they had developed resilience while learning to manage the illness, and if faced with challenges in the future would have the skills to cope. Some young people needed help with family issues. Others needed help with integrating back into school and dealing with the challenges of social interactions and social learning tasks of adolescence, particularly as they felt vulnerable and had lost some confidence.

### Use of Alternative Therapies

Seventy per cent of the young people used alternative therapies and these included 40 different types, some trying up to 10 different ones. The therapies ranged from naturopathy, chiropractics, homeopathy, Chinese herbs, intravenous vitamins, Reiki, Qi Gong, Tai Chi, Yoga, Myotherapy, Bowen therapy, massage, hypnosis, cupping, aromatherapy, color therapy, meditation etc. There were very few therapies that were considered of any value by the young people. The only ones that approached a 30% positive response involved massage (under a variety of different guises) for those with muscle pain and "good dietary advice" often in young people with associated abdominal discomfort. The most common comment was that they "wished their parents had not wasted their money or their energy by taking them to people who had promised to cure them but didn't."

### DISCUSSION

### Demographics and Baseline Symptoms

The finding of M:F ratio of 1:3 is consistent with other studies showing a consistently higher proportion of females to males with CFS, for example, Reyes et al. (39), Rowe et al. (40), and Bell et al. (7). The observation in this study that the ethnic background characteristics are not consistent with the demographics of the state, nor the clientele of other general medical or adolescent health clinics in the hospital, is interesting in the light of reports of CFS occurring in community surveys in Japan (41), Korea (42), and the United Kingdom (43). However, each of these studies was in adults and associated with high rates of psychological distress and based on survey information rather than a clinical assessment of CFS. There may be ethnic and cultural differences in how CFS is reported, for example in Japan it is rare for infection to be recognized as the trigger, and chronic sleep deprivation and stress are noted to be associated with the condition, where it is commonly described as school phobia (personal communication, Prof. Miike, 2008). Shi et al. (44) reported likely CFS from surveys of Chinese students associated with despondency and anxiety regarding school. There was a predominance of males. There are few documented pediatric studies in other ethnic populations. Due to the universal health care system and the demographic of other medical clinics in the hospital, access to health care is unlikely to be the main explanation. This cohort reported a high rate of post infective onset, as well as higher than expected rates of positive antinuclear antibody serology without evidence of clinical connective tissue diseases. It is recognized that people of northern European descent have a higher proportion of autoimmune diseases both in ethnic variation in expression of autoimmune diseases such as SLE (45) and the prevalence of multiple sclerosis (46).

Of note, was the consistency with which this cohort of young people responded regarding the severity and frequency of symptoms that was comparable to the earlier cohort that was obtained when there was very little awareness of the condition in young people (47). The high rate of reported infections at the onset and the consistency in responses suggests a relatively homogeneous group. The rates of anxiety and depression were also comparable with the previous sample and higher than the community sample. It is noted that the baseline rate of reported depression was only marginally higher than a large concurrent adolescent survey in the state (36). Crawley et al. (48) and a review by Lievesley et al. (49) have reported increased prevalence of anxiety and depression in young people with CFS implying that this association may be contributing to the morbidity.

### Follow Up Study

#### Outcomes

Follow up of this large well defined cohort with 87% of the study group providing data, has indicated that more than half the young people report recovery and many who do not report recovery, are also functioning well. The mean duration of illness was 5 years (range 1–15 years). By 10 years 68% reported recovery. The young people who reported recovery at 14 and 15 years could not identify any contributing factor. "It just happened." The duration of illness was comparable to the placebo arm in the 5-year follow up following the immunoglobulin trial and a mean of 8 years from onset (13). Sixteen percent were moderately unwell with 20% reporting "not quite back to normal" and the remaining 64% reporting recovery. Bell et al. (7) had a well characterized group also with a defined onset and likely post-infective, that was followed for approximately 13 years. Their findings were similar in that all had improved function and most considered that they had recovered except 20% remained unwell. Other follow up studies have been for short periods (1, 4, 6, 50); have not been diagnosed clinically (2); had half the cohort with fatigue of 1–6 months duration (4); the cohort was small in numbers (3); or the follow up did not have a high proportion participating (4, 5). Due to these differences, findings are difficult to compare. In this study, it is clear that there was variation in how young people defined recovery. There was a significant overlap in functional scores between those who had reported recovery and those that had not. In this study, as well as requesting an assessment as to whether they have recovered, an indication of their level of functioning that took into account stamina, recovery after activity, proportion of participation in work or school, whether the workload has been reduced, level of social contact and severity of symptoms. Early follow up questionnaires requested this in detail as well as using the Bell scale. Young people could consistently estimate their level of functioning. A global functional rating scale (**Appendix 3**) compiled from the surprisingly consistent descriptors of hundreds of young people with ME/CFS in the clinic has good inter-rater reliability. This however did not necessarily reflect their concept of whether they had recovered or not. They reported struggling with understanding what to expect as "normal functioning" as they had no frame of reference.

Other follow up studies have varied in how they have reported recovery making comparisons difficult. Differing measures such as measures of fatigue (8), symptom presence and functional outcomes, (9, 10), self-report (6) or a combination of global functioning and self-report have been used (7). Parslow et al. (11) have been investigating what aspects for defining recovery are important to young people with ME/CFS. Fatigue scales, Karnovsky scores, school attendance or physician's perspective (51) are useful markers of improvement but do not appear to capture the complexity of what is important to young people as well their tolerance level to the normal "ups and downs" of life and their satisfaction with what they are able to do.

In this follow up study there is a higher than expected rate of engagement in post-secondary education compared with the community rates, and a high proportion (95%) who are either working or studying part or full time. It was also noted that those who attended tertiary education, there was a much higher than community rate of completion of courses and lower than community rate of changing courses. In Victoria, there is a scheme available to apply for Special Entry Access to university if preparation for university entrance had been impacted by chronic illness, and usually very supportive Disability Liaison Units exist in universities to assist once enrolled. Universities recognize that young people who have faced adversity do not tend to waste their opportunities and their applications are usually given consideration.

It is noted that the proportion of young people with positive ANA is higher than expected without progressing to clinical autoimmune disease. There was a range of other illnesses reported which influenced the functional rating but were usually reported as separate from ME/CFS. Severe anxiety, depression, agoraphobia, eating disorder, although uncommon was linked with ongoing CFS. Presence of reported depression at follow up was linked with family discord or lack of support, difficulty with completing an education to equip for satisfying work, ongoing severe symptoms or disclosed history of sexual abuse that had often occurred after the onset of the illness.

### Feedback

Regular feedback has helped inform our management and advice that we can offer young people. There has been a clear message about how important it is to the young person to be believed, to feel supported and to be provided with a management plan that enables them to have some control of their lives. Maintaining social connectedness and support for continuing education is crucial to ensure their aspirations remain. Similar to the reported feedback from young people in the UK, advocacy for school and ability to continue with education was valued (11). The education system in the state allowed for flexibility in workload, school attendance, mix of Distance Education and school based learning as well as access to a Visiting Teacher service whose brief is to help liaise and coordinate the learning program at school and at home until the young person is well enough to return to school full time. Appropriate documentation to access these services is required but experienced Visiting Teachers explaining to other teachers significantly improved cooperation and satisfaction on the part of student and staff.

Anecdotal information about the perceived value of various alternative therapies has been appreciated by young people. As few therapies have even reached expected level for placebo intervention, the time and money saved has been often been used to support an interest area that has contributed to enriching their life.

Feedback indicated the importance of being sensitive about when psychological assistance is offered as there were many situations where they were not receptive until later in the illness. Sometimes this was related to an implied attitude that ME/CFS is a "psychological illness" rather than a chronic illness with its attendant challenges.

If graded exercise was implied as a means to recovery and symptoms were made worse, the young person often withdrew from any medical assistance and frequently from school. Some interpreted the message as indicating that the problem was with their cooperation or motivation, not with what was being advocated. Several were very angry and suspicious of medical profession. At other times, intervention programs were very helpful when self-management strategies such as organizational skills, sleep hygiene, advocacy for appropriate school program and appropriate strengthening exercises and advice regarding how to monitor increases in activity including "boom/bust planning."

Many reported that they appreciated the plan that recognized the impact of illness on every aspect of their life and the fact that they could reduce this effect by not neglecting these areas. This approach was not disregarding the fact that they were ill or implying that their belief that they were unwell was hampering their recovery. If they felt they were believed, they did not have to prove it, and they just needed to find a way to survive it in good shape and feel that they were supported in doing so. Families certainly indicated that they were grateful and much less anxious when there was a negotiated plan and they were supported in dealing with the education authorities. Similarly, the education authorities reported being grateful for information and support.

Although a variety of management strategies such as adaptive pacing, graded exercise, cognitive behavior therapy can be helpful (14), the specifics of what this entails and whether it is implemented as an individualized program is often not clear. However, Burgess et al. (52) found a family focused individualized home based rehabilitation program was well received for those with severe CFS. Evidence for significant improvement is hampered by difficulties in comparing outcome measures according to clinical presentation, patient characteristics, case criteria and degree of disability (16). The routine care that occurred in this cohort does include aspects of these strategies but as no two management plans were the same, the details of how they were implemented were not standardized. What did seem important was that the young person was devising the plan with the support of the family and cooperation of the school.

#### Outcomes and Baseline Characteristics

Although many reports (3, 43, 49, 53, 54) imply that the presence of depression or anxiety at the onset could potentially influence the recovery or function, there was no convincing evidence in this study that the presence or absence of depression, anxiety or illness severity was associated with outcomes in this cohort. Neither the presence of positive ANA titer nor documented EBV infection was associated with outcomes. Nonetheless, depression, anxiety or other illness documented at follow up was reported as affecting function and occasionally was linked with ongoing CFS or else reported as a separate issue following recovery. Therefore, this study was not able to identify any predictor for recovery.

### Strengths of This Study

The desire by young people to have answers to common questions ensured a high participation rate and frank responses from the large cohort over a long period of time. There was a very high proportion of the original cohort who willingly provided information despite the challenges in tracking such a mobile population. Despite little information in the public domain about the illness when some of the initial symptom inventory and background information data were collected, the consistency of responses indicates that the group is relatively homogeneous and consistent with earlier and later case definitions (19, 20, 35, 55) as well as being comparable to an earlier study group (12, 22). Although the comparison group in this cohort did not have comparable detailed baseline data nor the prospective repeated follow up questionnaires the 2 groups were only significantly different on age range and current education status but all other comparisons were comparable. Similarly, the concern that young people with severe CFS may not be represented in this study and therefore skew the findings, was not realized as some attended in wheel chairs and were clearly very limited, and the average functional score based on the Bell scale did indicate that at the beginning of the illness, they were severe.

The duration of illness and functional outcomes were also comparable between both groups, the placebo arm of the immunoglobulin trial as well as the Bell follow up study, which was a defined group for a prolonged period of time.

There was marked consistency in feedback comments. Regaining some control of their life by designing their own management plan was cited by the majority as the most helpful intervention.

### Weaknesses

This study has provided information from only one center and may not be representative of outcomes in another area of the country or internationally. The high rate of post-infective/defined onset CFS has indicated a relatively homogeneous group so that the outcomes may not be applicable to other groups. In addition although the management principles were similar, the actual plan was very individualized and the effective component would not be easy to measure. The timing of the various activity increases and the nature of these activities as well as the timing of psychological assistance was also very varied and individual, based on the young person's situation and readiness both from an illness point of view as well as developmental stage. Similarly, standardized outcome measures that have been used in other studies were not used consistently as the young people did not feel that they were asking questions that were important to them. Similarly school attendance, as such, was not a satisfactory measure of their educational progress but the number of subjects, workload and quality and quantity of task completion was often more satisfactory.

Early in this study it is likely that the presence and significance of orthostatic intolerance may have been missed, although it was noted that those with a slower onset of symptoms (over several months) frequently had associated hyperflexible joints.

Although the regular attempts at formal follow up ensured that the time of recovery was reported close to the event, the proportion of the current sample returned at each time period was not high. As these data were initially collected as part of routine clinical care rather than a formal investigation of duration and predictive factors, the baseline data was not complete. It does however appear that there was little difference in outcomes and other characteristics when both study group and comparison group were part of the final follow up study. So the concerns that lack of completion of baseline data may affect outcomes by potentially reflecting lack of engagement, severity of illness, or not disclosing mental health issues were unfounded.

### Further Study

In order to confirm if these estimates are able to be generalized, this study highlights the need for baseline questionnaire data that is not too onerous to be routinely collected in clinic cohorts. The data needs a severity or frequency scale, and an opportunity to indicate if the symptoms are currently not present, but were previously in the illness, as well as routine documentation of functional improvement and perception of recovery. Confirmation of the impact of depression and anxiety and illness severity at the outset and the perception that some of the contributing factors may be iatrogenic, and focusing on interventions that are important to young people would also be a fruitful area for further investigation. These factors may well vary depending on the sociocultural environment. The overlap between education and health and the importance of remaining engaged in education that was identified by young people as crucial for improved functional outcomes, as well as support and acceptance by family, medical personnel and schools and the importance of regular but not necessarily frequent follow up has been identified as areas that need further study. The impact of management of orthostatic intolerance on improving function warrants further study (17, 40). The suggestions that nonspecific autoimmune responses may be associated and that interventions such as intravenous immunoglobulin may be possible as a treatment option, warrants further investigation in a different cohort.

## CONCLUSIONS

Follow up of this cohort indicated that a significant proportion reported recovery and this was confirmed with reported participation in school, work or social activities as well as the global score. The mean duration of illness was 5 years with a range of 1–15 years. Other features of this cohort indicated that there was a high proportion of reported and documented infectious trigger, the most common being EBV. The reported symptoms were very consistent across the group. The ethnic background of the cohort was not representative of the population nor the hospital clientele, and as 17% reported a close family member who had had a prolonged recovery following an infectious illness, had diagnosed ME/CFS in the past or currently, a possible genetic predisposition is likely. Moderate to strongly positive ANA titers were more common than expected and not associated with any other clinical indication of connective tissue disorders. Depression rates were marginally above adolescent baseline rates in Victoria. Higher scores were commonly associated with severity of symptoms, "not being believed" or difficulty negotiating an appropriate program at school. Anhedonia symptoms were rarely reported and these were often when there was a recognized strong family history of depression. There was no difference in outcome rates with those with moderately severe depression at first visit and those with none, however depression was more commonly reported in those with reduced global scores at follow up and those who reported they had not recovered. Additional illnesses were relatively common at follow up and either distinct or comorbid with ME/CFS (Fibromyalgia, anxiety, depression, severe orthostatic intolerance, IBS, fructose/lactose intolerance). There were no suicides reported.

Young people reported that management strategies that allowed them some control back over their lives, that reduced the uncertainty for families and ensured that they received the appropriate understanding and support were the most valuable intervention. Symptom management, especially sleep and headache were very helpful. There were many comments about needing to be believed and understood by the medical profession, teaching profession and family. Comments about alternative therapies usually reflected their parent's anxiety and desire to find something that might help rather than being helpful to the young person.

Many young people reported that they could have improved their self-management as well as acknowledging that they could have been more prepared to accept some help regarding some of the social and psychological issues that they had to face as they were returning back into active life, including dealing with their lack of confidence regarding their ability to cope with adversity or acquisition of social skills due to their absences from school. They acknowledged that this was offered but they were not necessarily ready to access the help.

Remaining engaged in education and therefore their ability to pursue their aspirations was identified as crucially important but involved significant advocacy support and creativity to ensure this occurred. There was a high proportion who reported engaged in or completing post-secondary education (higher than national rate) and more than 95% were working or studying part or full time.

The answers to the common questions are that the majority recover (68% by 10 years). The mean duration was 5 years with range 1–15 years. The functional outcome is generally good, however the duration of illness over such a crucial period of development means that attention to the impact it has on education, social, emotional and physical development was identified by the young people as key to how they coped with, and survived the experience. There was no obvious predictor for recovery at the onset, but there are many helpful interventions including management plan, symptom management and remaining engaged in education. The level of maturity and resilience demonstrated in the young people in the feedback during the follow up was inspiring.

### REFERENCES

### AUTHOR CONTRIBUTIONS

The author confirms being the sole contributor of this work and has approved it for publication.

### ACKNOWLEDGMENTS

The willingness and frank responses by the young people and their families in participating in these studies is gratefully acknowledged. The detective skills of Judy Moon in contacting so many in the follow up study, the assistance of Tessa Brewin in coordinating the clinic in the early years and the input of Fran Bailey as Physiotherapist, helping with realistic exercises has been invaluable. The assistance with analyses in the earlier studies by my late husband Ken is gratefully acknowledged. The data collection, writing, analyses, and patient care was the responsibility of the author.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fped. 2019.00021/full#supplementary-material

chronic fatigue syndrome. Acta Paediatr. (2016) 105:e412–8. doi: 10.1111/apa. 13476

**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Rowe. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.


**4. Novel technologies for detecting the pathogens**

In recent years, the rapid detection of foodborne pathogens has developed rapidly. Molecular biology, nucleic acid hybridization, and other technologies have been highly valued and widely used in laboratory or factory production.

#### **4.1 Nanoparticles in pathogen detection**

Substances are manipulated at atomic, molecular, and supramolecular scales through nanotechnology ("nanotech"). Advances in manipulating these nanomaterials allow specific or non-specific binding of different biomolecules. The large specific surface area allows more biomolecules to be immobilized, thereby increasing the number of reaction sites that can be used to interact with the target species, which is one of the main advantages of biosensing using nanomaterials. In addition, nanomaterials have been widely used in 'label-free 'detection due to their excellent electronic and optical properties, and biosensors with enhanced sensitivity and improved response time have been developed [34].

Metal nanoparticles, especially gold and silver (5–110 nm in size) exhibit excellent properties, such as signal amplification, have potential application in various areas such as variable optical and electrical determinations. Gold nanoparticles (AuNPs) change the color aggregation from blue to red with the ability to scatter light, showing excellent chemical stability and electrical conductivity. AuNPs were used to detect Salmonella and *E. coli* O157: H7 organisms at 98.9 CFU/mL and 1–10 CFU/mL, respectively. Magnetic nanoparticles such as iron, nickel, and cobalt (size range of 1–100 nm) with electrical conductivity properties for utilization as a detection mean. Quantum dots (2–10 nm) were detected in *E. coli* O157:H7 103 CFU/mL through a semiconductor material consisting of semiconductor fluorescent nanonuclei (typically cadmium mixed with selenium or tellurium). Carbon nanotubes are formed by anisotropies of carboncontaining cylindrical graphene sheets. Multiwalled nanotubes (MWNTs, 2–100 nm) with photoluminescence and excellent electrical properties are composed of many concentrated single-walled nanotubes (SWNTs, 0.4–3 nm). A half conductance apparatus was used to monitor *E. coli* o157:h7 at 1 cell/mL restriction [35]. Thiol modified oligonucleotides covalently bound-based methods to gold nanoparticles are used as probes in various rapid detection ways. Due to its cost, functional chemistry is not so widespread. This method employs nonfunctional AuNPs to detect dsDNA and ssDNA [36].

#### **4.2 Polymerase chain reaction (PCR)**

Polymerase chain reaction (PCR) plays an important role in molecular methods in detecting foodborne pathogens. As early as 30 years ago, PCR, which was invented

## *Foodborne Pathogens of Enterobacteriaceae, Their Detection and Control DOI: http://dx.doi.org/10.5772/intechopen.102086*

for the detection of single bacterial pathogens present in food by identifying specific target DNA sequences [37]. PCR works by amplifying specific target DNA sequences in a three-step cycle [38]. Firstly, single-stranded DNA was obtained from target double-stranded DNA by high-temperature denaturation. Then, deoxyribonucleic acid was lead on the backbone of DNA by adding specific primers and heat-resistant DNA polymerase in the polymerization process of DNA, so a new double-stranded DNA was synthesized. The amplified products of PCR were stained by ethidium bromide on electrophoretic gels [39]. PCR such as loop-mediated isothermal amplification (LAMP), multiplex PCR (mPCR) and RT-PCR, etc. is used to detect foodborne pathogens, including *E. coli* 157: H7, *S. aureus*, *Campylobacter jejuni*, *Salmonella* and *Shigella* [40]. Because of the advantage of high specificity, efficiency and easy operation, LAMP and mPCR are used quite frequently [41–47].


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*2.5. cDNA Synthesis and Real Time q-PCR*

For each sample, 1000 ng of total RNA was retrotranscribed with an iScript™ cDNA Synthesis kit (Bio-Rad, Milan, Italy), following the manufacturer's instructions. The variations in the expression of five genes involved in stress response (*hsp26*, *hsp60*, *hsp70*, *COXI,* and *COXIII* [49]; see Supplementary Figure S1) were evaluated for adults. For nauplii, the variations in the expression of four other genes involved in developmental and differentiation processes (*HAD-like*, *tcp*, *UCP2,* and *CDC48*, [49]) were also tested (Supplementary Figure S1).

Undiluted cDNA was used as a template in a reaction containing a final concentration of 0.3 mM for each primer and 1 × SensiFASTTM SYBR Green master mix (total volume of 10 μL) (Meridiana Bioline). PCR amplifications were performed in AriaMx Real-Time PCR instrument (Agilent Technologies, Inc., Santa Clara, CA, USA), according to the manufacturer's instructions, using the following thermal profile: 95 ◦C for 10 min, one cycle for cDNA denaturation; 95 ◦C for 15 s and 60 ◦C for 1 min, 40 cycles for amplification; 95 ◦C for 15 s, one cycle for final elongation; and one cycle for melting curve analysis (from 60 ◦C to 95 ◦C) to verify the presence of a single product. Each assay included a no-template control for each primer pair. To capture intra-assay variability, all realtime qPCR reactions were carried out in triplicate. Fluorescence was measured using Agilent Aria 1.7 software (Agilent Technologies, Inc.). The relative expression ratios were calculated according to [50,51] using REST software (Version No., Relative Expression Software Tool, Weihenstephan, Germany; Equation (1)):

$$R = \frac{E\_{\text{target}}^{\Delta \text{C} \text{q target}} \left( {\text{Mean Control}} - {\text{Mean Sample}} \right)}{E\_{\text{refriger}}^{\Delta \text{C} \text{q reference} \ (\text{Mean Control} - \text{Mean Sample})}} \tag{1}$$

The expression of each gene was analyzed and internally normalized against *GAPDH* [49] using REST software (Relative Expression Software Tool, Weihenstephan, Germany) based on the Pfaffl method [50,51]. Relative expression ratios above 1.5 were considered as significant. The nonparametric Mann–Whitney test was applied to ΔCq (Cq gene of interest— Cq reference) values between treated and control samples (n = 3). *p*-Values < 0.05 were considered significant. Statistical analyses were performed using GraphPad Prism Software (version 8.02 for Windows, GraphPad Software, La Jolla, CA, USA, www.graphpad.com, accessed on 1 February 2021). Fold-change values were represented through a Heatmap generated by GraphPad Prism Software.


*5.1. Sigillography*

The lead seals preserved from the stage of Henry II prior to the events of Montiel represent him on the front under the traditional equestrian image, as Peter I had done, but it is striking how, after the fratricide and the definitive ascent of the Trastámaras to the throne, the new seals—of lead and measuring 51 mm in diameter—depicted him in

a majestic manner, seated upon two lions alluding to the Solomonic throne in order to manifest the king's authority to impart justice (Figure 3). His claim to succession was, thus, reaffirmed, as in the imago maiestatis the iura regalia are represented; that is, the most common symbols of power: a sword in his right hand, a crown and orb in his left, transmitting a sense of continuity along the dynastic line, and of legitimacy. The presence of the cross on the globe and the crown reiterates the idea of the king as Christ's envoy, while the fleur-de-lis topping the pommel of the sword reveals a French influence. On the reverse appear the heraldic arms of Castile and León. A fleur-de-lis cross creates the checkered pattern in which the castles and crowned rampant lions are alternately inserted. On the borders of both sides, we read the inscription: Ҡ S : ENRICI : DEI : GRACIA : REGIS : CASTELLE : ET : LEGIONIS" [27].

**Figure 3.** Seal of Henry II. Impression on lead. 1371. Archivo Municipal de Toledo. © Archivo Municipal, Toledo, Spain: (**a**) Front; (**b**) Back (The original document dates from 16 September, 1371, and is stored in the Archivo Municipal of Toledo (Spain), Secret Archive, Drawer 10, File 6, Number 6, Piece 3. See [28]).