The laboratory investigations performed prior to treatment did not uncover any concomitant disease. All laboratory tests for serum autoantibodies were negative, except for elevated titres of thyroid anti-microsomal antibodies in patient 1. All three patients had positive IgG, but negative IgM, to Epstein-Barr virus (EBV) and cytomegalovirus (CMV). Serologic tests for EBV and CMV, and in addition PCR analyses, were repeated during follow-up, without any evidence of active infection. The serum levels of IgG were slightly reduced from inclusion to the end of follow-up (12.1 - 5.8, 9.5 - 8.5, 12.5 - 11.6 g/L for patients 1, 2, and 3, respectively, normal range 6.0 – 15.3 g/L).

Patient 1

The patient with a transient improvement of symptoms during MIME chemotherapy for Hodgkin's disease had profound fatigue with little rest relief before rituximab infusion. She was mostly indoors and could only take walks for a few minutes, and had marked, diffuse muscle and skin pain. Sleep disturbances and cognitive dysfunction were profound. The first five weeks after rituximab she noted absolutely no change. From six weeks after treatment she started to experience increasing energy. The following weeks her condition improved substantially, she was able to take walks for up to two hours duration, and needed shorter periods of rest. Muscle and skin pain decreased markedly. Cognitive function improved substantially, and the sleep quality turned normal. However, from 14 weeks after the single rituximab infusion, her condition started to deteriorate, with gradually increasing fatigue, muscle and skin pain, cognitive dysfunction, and the sleep disturbances reappeared (Figure 1). Five months after the first rituximab infusion, she again had stable and disabling CFS symptoms, although she was still better than before the intervention. She insisted on retreatment and 20 weeks after her first treatment, she was given a new single infusion of rituximab at the same dosage. After six weeks, she again experienced a gradual recovery from all CFS symptoms (fatigue, pain, cognitive symptoms, sleep) with a major effect on quality of life. After the second infusion, the therapeutic effect was maximal at 12–16 weeks (32–36 weeks from the first infusion), with slow symptom worsening thereafter (Figure 1).

We then decided to start weekly oral methotrexate (Mtx) from 19 weeks after the second rituximab-infusion, at a time point when she did not have detectable CD19+ B-cells in peripheral blood. Mtx was started at one dose of 7,5 mg per week, and increasing to 12,5 mg per week during the next two months. From 10 weeks after onset of weekly Mtx, she has again experienced gradual recovery from CFS symptoms. She used Mtx for 25 weeks and reported significant improvement. Symptom recovery was slower with Mtx than rituximab. However, slow worsening resumed from 26 weeks after start of Mtx (Figure 1). She was again treated with a new (third) rituximab infusion at the same dosage, with ongoing weekly Mtx (35 weeks after start of Mtx treatment). At this time point B-cells in peripheral blood were increasing (Figure 1). In line with her experiences from the previous interventions, she had a major improvement of all CFS related symptoms, again starting from 6 weeks after the rituximab treatment (Figure 1).

Patient 2

A 34-year-old male encountered CFS in 2000 after infectious mononucleosis. At the same time, diabetes type I was diagnosed. In 2005 he was treated for a testicular seminoma Stage IIA with orchidectomy and postoperative radiation therapy and has since been recurrence free. Since 2000 he was completely unable to work. Most days he was only able to perform minor in-house tasks. He had severe general myalgic pain, headaches, chronic diarrhoea, increased sweating and dizziness. Cognitive function was markedly reduced and worsening over the years. He was unable to read more than a page or two of writing and suffered from hypersensitivity to noise.

Two weeks after single rituximab infusion (500 mg/m2) the diarrhoea vanished, while all the other symptoms remained unchanged. Before treatment he was mostly confined to rest indoors, with very brief walks, usually to and from the garden mailbox once daily. However, approaching six weeks after treatment he started to feel energy. During the next two weeks his activity level and muscle usage increased to the greatest level since the CFS debut. He could take one-hour walks and started to do carpentry on his house. Myalgic pain was markedly reduced. Cognitive functions improved remarkably, and he could now read a whole book without interruption. The hypersensitivity to noise decreased. He and his wife confirmed that family life had improved considerably.

The clinical benefit was most pronounced until 12 weeks after the rituximab infusion, thereafter all CFS symptoms started to gradually incline. At 18 weeks after the intervention, he described symptoms as almost equal to baseline (Figure 1). He was then retreated with two infusions of rituximab 1000 mg, given two weeks apart. As following the first treatment, he started to recover first from diarrhoea (after 3 weeks). Then, after six weeks, he reported less cognitive symptoms and days later the fatigue started to improve.

The double rituximab infusion gave a clear CFS symptom improvement most prominent 16 weeks after the infusion. Thereafter, he has experienced a very slow increase in symptoms. However, six months after the infusion, he still had some clinical response. Fifty weeks after the first rituximab infusion (30 weeks after the second) he started treatment with weekly oral Mtx. At the time points of the second rituximab treatment (week 19) and of Mtx initiation (week 50) he did not have detectable CD19+ B-lymphocytes in peripheral blood. After 12 weeks on weekly oral Mtx (62 weeks after first intervention) he still did not experience symptom amelioration, and was then given a new double rituximab infusion (each of 1000 mg) two weeks apart, with ongoing Mtx treatment. At this time the B-cell number increased (Figure 1). In accordance with the previous courses, he after eight weeks (week 70) again had a marked improvement of fatigue, cognitive function, muscle pain, dizziness, noise intolerance, sweating and diarrhoea (Figure 1).

Patient 3

The 23-year-old school pupil attained CFS following infectious mononucleosis in 2001. School activities had slowed to the extent that she was in her sixth year of a three-year program. Marked reduction in physical activity and only moderate relief from rest, diffuse muscle pain and diarrhoea were present. Cognitive dysfunction was moderate. She felt frustrated as a consequence of the disease and its negative impact on her studies and social life.

Between five and six weeks after treatment she felt more energized, especially in short-lasting bursts. Myalgic pain was slightly improved, resulting in reduction in paracetamol usage. The diarrhoea terminated. She had less fatigue after special occasions with exertion, but did not experience significantly increased energy in daily life and did not manage to increase efforts at school. From 13 weeks after infusion she had stable CFS symptoms, only slightly better than at baseline (Figure 1).

Then, from 26 weeks after the infusion, she experienced a dramatic recovery of all CFS symptoms over the next few weeks. Fatigue improved to the extent that she could participate fully in school education, she resumed social life, started more extensive physical exertion including jogging and needed no rest during daytime. All muscle pain vanished, she had better appetite, stools normalized, and she also had regular menstrual cycles for the first time in 7 years. Concentration ability and memory improved substantially. There were no other interventions that could explain her symptom recovery. This major CFS improvement lasted from 6 months until 10 1/2 months after rituximab infusion. She then experienced gradual symptom worsening over the next 4 weeks, with increasing muscle pain, relapse of fatigue, cognitive dysfunction and loose stools. She was then (48 weeks after the first rituximab) treated with two infusions of rituximab (500 mg/m2) given two weeks apart (Figure 1). At this time point B-lymphocytes in peripheral blood had recovered. From 5 weeks after the second rituximab treatment, she again experienced short bursts of being energized, and from 10 weeks she had slight improvement of muscle pain and cognitive function. Almost identical to the symptom course after her first intervention, from approximately 5 months after the second (double) rituximab treatment she experienced a dramatic response on all CFS symptoms, to the same extent as described above (Figure 1).

Toxicity

No acute toxicity was seen. Patients 1 had two, and patient 2 one uncomplicated upper respiratory tract infection, all appearing approximately two months after one of the rituximab treatments. Patient 1 had an episode of localized herpes zoster 21 weeks after the first rituximab infusion. No other toxicity was noticed during follow-up.