In this study the positivity of BDV CIC was detected in 36.6% of addicted patients at the beginning of detoxification, in 42.9% after eight weeks of abstinence and in 37.3% of the control group of blood donors.
Surprisingly, we did not find a higher BDV CIC positivity in patients with alcohol and drug dependence in comparison with healthy individuals. Nor did we find any significant difference between BDV CIC positivity at the beginning of detoxification and after eight weeks of abstinence. A BDV CIC positivity was not associated with the premature ending of treatment. We cannot compare our results with other studies because no other studies of BDV infection in addicted patients have been published. Larger studies are needed to confirm these results.
We assumed a higher positivity of BDV infection in addicted patients because of the higher BDV CIC positivity found in psychiatric patients in the Czech Republic, and possible changes in immunity and close relationships with endemic regions for BDV in central Europe. The positivity of BDV CIC in addicted patients in the present study was lower than that found in our previous study in psychiatric patients (with psychotic and affective disorders). We detected BDV CIC positivity in 66.7% of psychiatric patients and the intensity of BDV infection was positively correlated with psychopathology .
Bode and colleagues detected a BDV CIC positivity of between 40% and 50% in psychiatric patients and between 90% and 100% in patients with an acute state of affective disorders in comparison with a positivity of 30% in blood donors. The persistence of high amounts of plasma BDV CIC and Ag correlated with the severity of depression .
Nunes and colleagues examined BDV RNA by using reverse transcriptase polymerase chain reaction in psychiatric patients, their relatives and healthy controls. Borna disease virus RNA positivity was detected in patients with psychotic disorders (44.4%), relatives without mental disorders (50%), relatives with mental disorders (37.5%) and healthy controls (14.8%) .
Another study failed to detect BDV positivity in psychiatric patients (depression, bipolar disorders, schizophrenia) where neither the BDV antibody nor the BDV RNA was proven in psychiatric patients or healthy controls . The authors of this study suggested that BDV infection might not be associated with mental disorders in this region.
We examined the presence of BDV CIC in the serum by using the ELISA method and were able to detect active BDV infection. The ELISA method finds a higher BDV positivity than other laboratory methods (IFA) [4, 5].
Borna disease virus infection (antibodies) was first detected using serological methods, especially immunofluorescence (IFA), by Rott and colleagues . A positivity of BDV Ab was found of between 1% and 4%. Another laboratory method which has been used for the detection of BDV Ab is western blot (WB). These methods detecting only BDV Ab were found to be less sensitive than the ELISA method and were not able to detect acute phases of BDV infection. Antibodies, when found alone, can indicate a previous contact with BDV but not an acute state. Bode and colleagues examined plasma samples using ELISA for BDV CIC positivity and immunofluorescence for the detection of antibodies. The BDV CIC determination found a higher prevalence of infection than previous IFA methods (ten times higher infection rates) [4, 26].
Antigenaemia indicates an acute and productive phase of infection. During this phase of BDV infection antibodies bind to the antigens and form circulating immunocomplexes (CIC), which are measurable for weeks or months. The frequency and stability of BDV CIC makes them available screening markers of BDV infection, as recommended by some authors. By using the ELISA method it is possible to detect viral Ag in plasma. A higher positivity of BDV Ag was detected in patients with depression and the intensity and duration of antigenaemia was correlated with the severity of symptoms [4, 5]. The disadvantage of this detection method is the very short period of antigaenemia in the acute phase of BDV infection [4, 5, 26].
Several authors used the detection of viral RNA in PBMCs (peripheral blood mononuclear cells) or brain tissue by polymerase chain reaction (PCR) for the diagnosis of BDV infection. However, other researchers did not use this method for the diagnosis of BDV infection because of the possibility of sample contamination during the laboratory procedures , although this contamination should not occur when the detection of BDV RNA is performed according to international security instructions . The absence of BDV RNA in the serum cannot exclude the presence of infection as the low amount of RNA is not possible to detect using this method. The second reason for not using BDV RNA detection is that that the presence of BDV RNA does not reflect an active state of viral replication .
A limitation of our study was that only BDV CIC positivity was detected. Antigens and circulating immunocomplexes are the markers of BDV infection activity. Some CIC-negative patients can have acute viraemia and are only Ag positive. Furthermore, the immune impairment in addicted patients lowers the ability to produce Ab and provides far less CIC formation. Also, immune changes in addicts are not important for BDV infection.
We found a higher BDV CIC positivity in younger patients and a decrease in BDV CIC positivity with the increasing age of patients. This finding is consistent with the observations of the Polish psychiatric population  and German  and Italian [29, 30] studies which demonstrated a significantly higher BDV CIC positivity in children. Patti and colleagues investigated BDV positivity in children in Italy. A BDV CIC positivity was found in 57%. The prevalence of BDV infection was found to be higher in children, particularly in the third year of life, then, it decreased until 15 years of age, where another increase was observed [29, 30]. Scholbach and colleagues demonstrated a higher BDV Ag and CIC positivity in children. There were two age intervals of peak BDV positivity, the first with a peak at 6 months old and the second with a peak value around 2-3 years old. These findings supported the possibility of vertical transmission of BDV infection. Also, children of 2-3 years old are likely to be in more intensive contact with the secretions of animals, which are associated with a greater possibility of BDV transmission in children than in adults .
Flower and colleagues found a positive association between BDV positivity and elevated liver enzymes. They described the association between BDV antigenaemia and elevated plasma levels of ALT and GGT in multi-transfused patients. It was not clearly explained whether these findings had any causal associations . In our study we detected the opposite situation, where a higher BDV CIC positivity was significantly associated with a lower level of GGT, implying milder liver impairment. We are not able to satisfactorily explain this finding yet. The influence of a lower age (i.e. a shorter history of abuse and/or higher frequency of non-alcoholic drug abuse) was not confirmed.
Alcohol and drug abuse interferes with humoral and cellular immunity and leads to the suppression of human resistance to bacterial and viral infections, increasing infection susceptibility. This direct influence on immunity in combination with other risk factors (risk behaviour, vitamine deficiency) is associated with impairment of the immune system [22, 23].
We proved no significant association between BDV CIC positivity and the history of infectious diseases (hepatitis C) or the presence of infection at the time of blood sampling. The presence of other infections can reactivate the latent BDV infection and cause the increase of BDV positivity [2, 3]. Cotto and colleagues investigated the presence of BDV infection (BDV RNA) in two groups of immunocompromised patients (with HIV infection and treated with immunosuppressive medication). They detected a significantly higher BDV positivity in patients with HIV infection compared with the second group and healthy controls .
In our study, contact with animals was not associated with BDV CIC positivity, although the majority of the addicted patients from our study had been exposed to animals (especially pets such as dogs and cats).
Some studies described a higher BDV positivity in people who were in contact with infected animals. Weisman and colleagues reported a significantly higher positivity of BDV antibodies (46%) in workers exposed to infected ostriches versus a BDV positivity of 10% in controls. There was a strong correlation between the intensity of exposure and the rate of seropositivity . Takahashi and colleagues found a significantly higher seropositivity of BDV (from 2.6% to 14.8%) in blood donors from regions where horse farms were concentrated compared with a BDV positivity of 1% in blood donors from other regions . These findings support the possible animal-to-human transmission of BDV infection. In contrast, another study from Bangladesh did not confirm this hypothesis. The authors surveyed horses and their caretakers for BDV antibody positivity and found a BDV positivity of between 25-30% in the horses but none of caretakers were positive for BDV . Thomas and colleagues reported that people working or living on livestock farms had a higher BDV seroprevalence compared with other farms. Exposure to animals did not increase the risk of BDV positivity .
Another explanation for not finding an association between contact with animals and BDV CIC positivity is that these results might have been derived from an area in which the BDV infection of animals is not endemic. We still have no data about the prevalence of BDV infection in animals in the Czech Republic. Several studies found a high BDV positivity in horses and sheep [1, 36] and a lower BDV positivity in other species (dogs, cats, cattle) . Addicted patients from our study were frequently exposed to pets such as cats and dogs (90.2%) but they were not exposed to farm animals, which we could expect to have a higher BDV positivity than pets.