3.1. HHV-6 and B-19 Serology
Specific anti-HHV-6 antibodies were detected in 87/108 (80.6%) plasma samples (IgG-71, IgM-3, IgM + IgG-13) from the ME/CFS patients versus 69/90 (76.7%) practically healthy persons' plasma samples (IgG-67, IgM + IgG-2) and specific anti-B19 antibodies in 92/108 (85.2%) plasma samples (IgG-62, IgM-6, IgM + IgG-24) from the ME/CFS patients versus 55/90 (61.1%) plasma samples ( IgG-44, IgM-2, IgM + IgG-9) from the practically healthy persons.
3.2. Prevalence of Active HHV-6, HHV-7, and B19 Infections
Active viral infection/coinfection was detected in 70 (64.8%) and latent—in 32 (29.6%) out of 108 patients with ME/CFS. Six (5.6%) patients were negative for viral infection (). The rate of active viral infection was significantly higher in patients comparing to the rate in the practically healthy persons (70/108 and 12/90, resp.; Odds ratio 0.14, 95% CI 0.07–0.26, P = 0.0001). In the patients significant difference was detected between the frequency of single active and concurrent active viral infection (41/70, 58.6% and 29/70, 41.4%, resp.; Odds ratio 2.0, 95% CI 1.02–3.92, P = 0.044). Among the patients who were infected with a single virus, the rate of HHV-7 active infection (40%) was significantly higher in comparison with B19 (15.7%, Odds ratio 3.58, 95% CI 1.60–7.97, P = 0.002) and HHV-6 active infection (2.9%, Odds ratio 22.7, 95% CI 5.13–100.1, P < 0.0001). No significant difference was detected between the frequency of active concurrent dual HHV-6 + HHV-7 and dual HHV-7 + B19 infection (Odds ratio 0.61, 95% CI 0.25–1.47, P = 0.273).
Frequency of Active HHV-6, HHV-7 and B19 Infection in ME/CFS Patients and Practically Health Persons.
HHV-6B was identified in 15 and HHV-6A in one out of 16
PBL and plasma DNA samples.
3.3. HHV-6 DNA Load in PBL DNA Samples
The number of HHV-6 DNA copies in PBL DNA of the ME/CFS patients with and without HHV-6 viremia was compared. A clear increase of HHV-6 load in PBL DNA was detected in 16 patients with plasma viremia in comparison with seven patients without it (132.61 ± 41.38 × 103
and 8.73 ± 3.96 × 103
g DNA, resp.).
3.4. Relationship between Plasma Level of TNF-α, IL-6, and IL-4 and Active Viral Infection/Coinfection
To investigate the relationship between the active viral infections and plasma cytokine levels, the levels of proinflammatory (TNF-α
, IL-6) and anti-inflammatory (IL-4) cytokines were measured in 106
ME/CFS patients (). The mean levels of TNF-α
and IL-6 cytokines were significantly higher in patients with active viral infection/coinfection (52.51 ± 15.13
pg/mL, 18.59 ± 3.56
pg/mL, resp.) than in those with latent (18.81 ± 2.52
pg/mL, 2.56 ± 1.02
pg/mL, resp.; P
< 0.0001) or without viral infections (7.71 ± 3.07
pg/mL, 1.32 ± 3.07
pg/mL, resp.; P
< 0.0001). No significant difference was detected between expression levels of TNF-α
in the patients with active single HHV-7 and active single B19 infection, as well as with dual active HHV-6 + HHV-7 and dual active HHV-7 + B19 coinfection ().
Plasma cytokine levels in patients with ME/CFS.
The highest level of TNF-α
was detected in patients with active triple HHV-6+HHV-7 + B19 coinfection. Significantly higher level of IL-6 expression was detected in plasma samples of the patients with single active B19 infection in comparison with the patients with single active HHV-7 infection (P
< 0.001) (). The mean levels of this cytokine were also significantly higher in patients with active concurrent HHV-7 + B19, as well as in patients with triple HHV-6 + HHV-7 + B19 infection (29.19 ± 6.26
< 0.001) in comparison with the level in patients with dual HHV-6 + HHV-7 infection (11.22 ± 3.14
pg/mL). None of the ME/CFS patients had increased plasma level of IL-4.
3.5. Assessment of Active Betaherpesviruses and B19 Infection/Coinfection in Association with Clinical Outcomes in ME/CFS Patients
Severe chronic fatigue for at least six months or longer was observed in all patients irrespective of the causation of the active infection (total FSS scores 58.89–60.99, P < 0.05). Subfebrility, tender cervical or axillary lymph nodes, and postexertional malaise were not revealed in the patients with single B19 active infection but were detected in patients with single HHV-7 active (50%, 75%, 100%, resp.), dual HHV-6 + HHV-7 (70%, 80%, 90%, resp.) as well as triple HHV-6 + HHV-7 + B19 coinfection (74.1%, 68.4%, 74.1%) ().
Symptoms of ME/CFS in patients with active viral infection/coinfection.
Although muscle pain was observed in all patients, the frequency of multijoint pain was more clearly displayed in all patients with active B19 infection, as in cases of single as well as in cases of coinfection with β-herpesviruses.
Severe postexertional malaise corresponding to “Exercise brings on my fatigue” by FSS was detected in all patients (mean score 6.94 ± 0.243 from 7 maximum) with single HHV-6, HHV-7, and in 9/10 with dual HHV-6 + HHV-7 coinfection as well as in 14/19 with triple HHV-6 + HHV-7 + B19 coinfection (90% and 74%, resp.).
Neuropsychological disturbances were observed in all 70 patients. Impaired memory was detected in 22 out of 57 (38.6%) patients with active β-herpesviruses infection/coinfection but not observed in patients with single HHV-6 and single B19 infection. Impaired concentration was detected in 34 out of 70 patients (48.6%), more frequently in patients with B19 infection. Sleep disturbances were revealed in 49 out of 70 (70%) patients, the sleepiness was more characteristic for patients with single HHV-6, B19, and dual HHV-7+B19 coinfection (2/2, 11/11 and 15/15, resp.).
Headaches of new type were observed in 16 out of 30 (53.3%) patients with B19 infection/coinfection and in 14 out of 38 (36.8%) patients with HHV-7 and dual HHV-6 + HHV-7 infection.
Chronic fatigue (for at least 6 months or longer period) was observed also in all 38 patients with latent infection and without infection (32 with latent infection and 6 without infection). Postexertional malaise (23/38, 60.5%, mean score 5.23 ± 0.135 from 7 maximum), impaired memory (34/38, 89.5%), decreased concentration (32/38, 84.2%), and sleep disturbances (24/38, 63.2%) were predominant symptoms in these patients. Subfebrility (10/38, 26.3%) and lymphadenopathy (11/38, 29%) were observed only in patients with latent single HHV-7 and dual HHV-6 + HHV-7 infection/coinfection; muscle pain (14/38, 36.8%) in patients with β-herpesviruses infection/coinfection (11/14) as well as with B19 infection/coinfection (3/14). Multijoint pain was observed in 15 out of 38 (39.5%) patients and in 10 of them B19 infection/coinfection was found. Headaches of new type were observed in 13 out of 38 (34.2%) patients. Among these 13 patients β-herpesviruses infection/coinfection was detected in 9 patients and B19 infection/coinfection in 4 patients. Clinical manifestations of the above-mentioned symptoms were not severe (total FSS scores were 42.83–48.90, P < 0.05).
Subfebrility, lymphadenopathy, malaise after exertion, muscle pain, multijoint pain, sleep disturbances, and headaches of new type were more frequent in patients with active viral infection/coinfection than in patients with latent infection and without infection. Whereas presence of impaired memory and impaired concentration was more frequent in patients with latent infection and without infection in comparison to the patients with active infection.