Helena Dahl

Polymerase chain reaction on cerebrospinal fluid for diagnosis of virus-associated opportunistic diseases of the central nervous system in HIV-infected patients

Objective:To assess the diagnostic reliability of polymerase chain reaction (PCR) on cerebrospinal fluid (CSF) for virus-associated opportunistic diseases of the central nervous system (CNS) in HIV-infected patients. Design:CSF samples from 500 patients with HIV infection and CNS symptoms were examined by PCR. In 219 patients the PCR results were compared with CNS histological findings. Methods:Nested PCR for detection of herpes simplex virus (HSV) type 1 or 2, varicella zoster virus (VZV), cytomegalovirus (CMV), Epstein–Barr virus (EBV), human herpesvirus 6 (HHV-6), and JC virus (JCV) DNA. Histopathological examination of CNS tissue obtained at autopsy or on brain biopsy. Results:DNA of one or more viruses was found in CSF in 181 out of 500 patients (36%; HSV-1 2%, HSV-2 1%, VZV 3%, CMV 16%, EBV 12%, HHV-6 2%, and JCV 9%). Among the 219 patients with histological CNS examination, HSV-1 or 2 was detected in CSF in all six patients (100%) with HSV infection of the CNS, CMV in 37 out of 45 (82%) with CMV infection of the CNS, EBV in 35 out of 36 (97%) with primary CNS lymphoma, JCV in 28 out of 39 (72%) with progressive multifocal leukoencephalopathy. Furthermore, HSV-1 was found in one, VZV in four, CMV in three, EBV in three, HHV-6 in seven, and JCV in one patient without histological evidence of the corresponding CNS disease. Conclusions:CSF PCR has great relevance for diagnosis of virus-related opportunistic CNS diseases in HIV-infected patients as demonstrated by its high sensitivity, specificity, and the frequency of positive findings.

Absence of seven human herpesviruses, including HHV-6, by polymerase chain reaction in CSF and blood from patients with multiple sclerosis and optic neuritis

Several members of the herpesvirus family have been implicated in the pathogenesis of multiple sclerosis (MS). Recently, HHV‐6 viral antigen has been demonstrated in association to MS plaques, as well as DNA from human herpesvirus 6 (HHV‐6) in cerebrospinal fluid from a few MS patients by polymerase chain reaction (PCR). In the present study, CSF from patients with MS, optic neuritis and other neurological diseases, as well as consecutive CSF and serum samples from MS patients included in a clinical trial with acyclovir, were analysed by nested PCR for the presence of DNA from herpes simplex virus 1 and 2, Epstein‐Barr virus, varicella zoster virus, cytomegalovirus, human herpesvirus 6 and 7. No virus DNA was found in any CSF (n= 115) or serum (n= 116) sample. These findings argue against a continuous disseminated herpesvirus infection in MS, but do not rule out a lesion‐associated, low‐grade herpesvirus infection within the MS brain.

IgG antibodies to human herpesvirus-6 in children and adults both in primary Epstein-Barr virus and cytomegalovirus infections

Antibody titers against human herpesvirus-6 (HHV-6) were determined in 80 healthy adults and 100 children and teenagers from Sweden to gain information on the role of the virus and its epidemiology. Based on a positive immunofluorescence titer of 1:10 and above, about 85% of the adults and children were seropositive with 60% seropositivity of children below age one year. Titers were generally higher in patients with simultaneous EBV or CMV infection, yet crossreactivity appeared essentially no problem. HHV-6 thus is ubiquitous like other herpesviruses. Primary infection seems to occur early in life, and reactivation or delayed primary infection may be associated with a variety of disorders.

Reactivation of human herpesvirus 6 during pregnancy.

Reactivation of human herpesvirus 6 (HHV-6) and cytomegalovirus (CMV) during pregnancy and transmission of the viruses to the fetus were investigated by polymerase chain reaction (PCR) and serology. In all, 104 blood samples were obtained 3 times during pregnancy and once at delivery. In another 107 women, samples were obtained only at delivery. Cord blood samples were obtained from both groups of women. HHV-6 DNA was detected in 41%-44% of the samples during months 3-8 of pregnancy, in 25% at delivery, and in 24% of age-matched controls. HHV-6 DNA was found in 1.0% of the cord blood samples. CMV DNA was detected in 1.7% of leukocytes from 104 pregnant women but in no cord blood sample. IgG antibodies to HHV-6 were found in 96% and CMV IgG in 62.5% of the women. HHV-6 IgG titers were significantly higher in HHV-6 PCR-positive women. Thus, HHV-6 reactivation seems common during pregnancy, and transfer of HHV-6 to the fetus may occur in approximately 1% of pregnancies.

Epstein‐Barr virus DNA in serum after liver transplantation ‐ surveillance of viral activity during treatment with different immunosuppressive agents

In immunocompromised HIV-infected and transplanted patients, there is a risk of developing Epstein-Barr virus (EBV)-associated lymphoproliferative disorders (LPD) and lymphomas. EBV has previously been detected by the polymerase chain reaction (PCR) in cerebrospinal fluid from all AIDS patients with EBV-associated cerebral lymphomas. We therefore thought it would be of interest to determine whether transplant patients with extracerebral EBV-associated LPD have detectable EBV genomes in serum. Nested PCR (nPCR) showed that 58% (18/31) of liver transplant (LTX) patients had EBV DNA in 17% (21/125) of serum samples obtained within the first 3 months after LTX. In 39% (7/18) of the patients, the first EBV nPCR-positive sample was found within 2 weeks post-LTX. Basic immunosuppression with cyclosporin A or FK506 did not seem to influence the frequency of detectable EBV genomes in serum. In contrast, positive EBV nPCR correlated to secondary OKT3 treatment for severe acute rejection (P=0.009). EBV-associated malignant lymphoma developed in three patients 2–6 months post-LTX. In all of them, EBV DNA was amplifiable within 12–14 days after LTX. The EBV antibody titers were not directly related to detectable EBV DNA in serum. We conclude that monitoring of LTX patients receiving increased immunosuppression by nPCR for EBV DNA in serum may help in the early identification of those at risk of developing EBV-associated LPD.

An enzyme-linked immunosorbent assay for IgG antibodies to human herpes virus 6

An indirect enzyme-linked immunosorbent assay (ELISA) with human herpes virus 6 (HHV6) membrane antigen was compared with indirect immunofluorescence assay (IFA) for measurement of HHV6 IgG antibodies. Five hundred serum samples from 403 Swedish patients with suspected symptomatic Epstein-Barr virus (EBV) infections were examined. The specificity of the ELISA compared with IFA was 98.7% and the sensitivity was 98.4%. In 90% of the patients, IgG antibodies to HHV6 were detected with both assays. The highest HHV6 IgG titers were found mainly in patients with EBV or CMV infections, but HHV6 mononucleosis was not diagnosed. The same HHV6 antigen was assessed for IgM ELISA but was found to be of limited value due to high IgM reactivity with the control antigen. The HHV6 IgM ELISA requires further investigation. The IgG ELISA described is a reliable alternative to IFA for measurement of HHV6 IgG antibodies and for large scale epidemiological studies.

Lymphoproliferative responses to human herpesvirus-6 variant A and variant B in healthy adults.

Human herpesviurs‐6 (HHV‐6) isolates can be classified into variants A and B, and over 95% of people older than 2 years of age are seropositive for either or both variants. However, the prevalence of the two HHV‐6 variants is still not defined since the serological methods used at present cannot discriminate one variant from the other. Lymphoproliferative responses to glycine extracted cellular antigens from human herpesvirus‐6 (HHV‐6) GS strain (variant A)‐ and Z 29 strain (variant B)‐infected T‐lymphoid cell lines were examined in healthy Swedish and Japanese adults. Nine of 36 (25%) persons had responses to the GS antigen, while 21/36 (58%) had responses to the Z 29 antigen (P = 0.008). Individuals with low anti–HHV‐6 IgG titers (⩽320) were more likely to respond to the Z 29 antigen than to the GS antigen (P = 0.006), while there was no difference in those with high anti–HHV‐6 IgG titers (⩾1280). Three of 7 Japanese adults had lymphoproliferative responses to the GS antigen compared with 6/29 Swedes (not significant), and 7/7 Japanese had lymphoproliferative responses to the Z 29 antigen compared with 14/29 Swedes (P = 0.03). Lymphoproliferative responses were neither related with the presence of HHV‐6 DNA nor related with the presence of HHV‐7 DNA in peripheral blood cells. These results suggest a higher prevalence of HHV‐6 variant B than variant A in both Swedes and Japanese adults, and possibly a difference in either the HHV‐6 virus strains and/or the nature of immune response of Swede and Japanese. J. Med. Virol. 57:134–139, 1999.

Human herpesvirus 6 in cerebrospinal fluid of patients infected with HIV: frequency and clinical significance

The objective was to evaluate the frequency of human herpesvirus 6 (HHV-6) DNA detection in the CSF of patients infected with HIV and its relation to brain disease and systemic HHV-6 infection.  Nested polymerase chain reaction (PCR) was used to analyse CSF samples from 365 consecutive HIV infected patients with neurological symptoms. When available, plasma and brain tissues from patients whose CSF was HHV-6 positive were also studied.  HHV-6 was found in the CSF of eight of the 365 patients (2.2%): two had type A and four type B; the HHV-6 variant could not be defined in the remaining two. All eight patients had neurological symptoms and signs related to concomitant opportunistic brain diseases, including cytomegalovirus (CMV) encephalitis in five patients whose CSF was also positive for CMV-DNA. Opportunistic infections but no other unexplained lesions were also found in the brain of all of the four patients who underwent neuropathological examination. Both HHV-6 and CMV were also detected in the plasma of respectively five and seven of seven patients whose CSF was HHV-6 positive.  In conclusion, HHV-6 type A or B DNA was infrequently found in the CSF of HIV infected patients, in association with both CMV brain infection and systemic HHV-6 replication. However, no certain relation between HHV-6 and brain disease was found.

Diagnosis of Pulmonary Infections in Immunocompromised Patients by Fiber-optic Bronchoscopy with Bronchoalveolar Lavage and Serology

Fiber-optic bronchoscopy (FOB) and bronchoalveolar lavage (BAL) were performed on 67 occasions in 57 immunocompromised patients with symptoms consistent with pulmonary infection. Diagnosis was achieved more often in renal transplant patients than in patients with hematological malignancies (85% versus 28%). Culture (bacteria, virus, fungi), staining and microscopy (bacteria, fungi, Pneumocystis carinii (PC)) and antigen detection by indirect immunofluorescence (cytomegalovirus (CMV), respiratory viruses, PC, Legionella) were used for diagnosis. On 20 occasions transbronchial biopsies with histopathologic examination were performed. In addition, serology comprising the herpes group (HHV-6) and respiratory viruses was done. A microbial diagnosis was obtained on 45% of occasions. The most common pathogens found were CMV (31%) and PC (25%). On 22 (33%) occasions a rapid diagnosis of 1 or more microbial agents was obtained within 24 h by conventional staining or indirect immunofluorescence. The clinical relevance of findings of CMV, HHV-6, and Epstein-Barr virus in BAL by polymerase chain detection on 18, 6 and 3 occasions is discussed. On 4 occasions pathogenic bacteria were found. It was not possible to relate findings of coagulase-negative staphylococci, alpha-streptococci and Candida albicans to the pulmonary infection.

In vitro inhibition of SARS virus replication by human interferons.

Four different types of human interferon, interferon-β (IFN-β), recombinant IFN-α2a and IFN-α2b and natural IFN-α were tested for antiviral activity against SARS-coronavirus. The experiments were performed using in vitro cultivated monkey Vero E6 cells. IFN-β was found to be the most highly active antiviral agent, followed by natural IFN-α, whereas the 2 recombinant IFN-α2 species were poorly active in the system used. These results suggest that IFN-β as well as natural IFN-α may be used for the treatment of SARS.