Seroprevalence of HHV 8 antibodies among the general population and HIV positive persons in the Czech Republic.

BACKGROUND: The seroprevalence rates of herpesvirus 8 (HHV 8) antibodies were determined for the general Czech population and HIV-positive individuals. OBJECTIVES: Six hundred and sixty six serum samples from the general Czech population and 129 serum samples from HIV-positive persons were tested for the presence of antibodies to the HHV 8 lytic and latent antigens. STUDY DESIGN: HHV 8 antibodies were detected by the indirect immunofluorescence test. RESULTS: In the general Czech population, only 2.4 and 0.3% of the serum samples tested positive for antibodies against the lytic and latent HHV 8 antigens, respectively. As many as 34.9 and 10.9% HIV positive individuals had antibodies to the HHV 8 antigens, respectively. Only three of them have developed Kaposi's sarcoma (KS) to date. At the time of KS diagnosis, the three patients had antibodies to both HHV 8 antigens. HIV-positive homo/bisexuals were at significantly higher risk of acquiring HHV 8 infection compared with HIV-positive heterosexuals. The increase in HHV 8 seroprevalence was associated with progression of the HIV infection from stage A to stage B. No correlation was found between the HHV 8 seroprevalence and CD 4+T-lymphocytes counts or the HIV viral load. CONCLUSIONS: Among the general Czech population, the HHV 8 seroprevalence is as low as in the West European countries. The mean HHV 8 seroprevalence rate in HIV-positive individuals was 34.9% and was comparable with those reported in other low seroprevalence countries.

Presence of herpes simplex virus (HSV) in peripheral leukocytes of patient who developed active HSV infection after bone marrow transplantation.

BACKGROUND: Despite of prophylactic antiviral therapy, latent HSV may be reactivated in bone marrow transplant (BMT) recipients and cause serious disease. Rapid diagnosis of HSV infection is needed to prompt institution of appropriate therapy. OBJECTIVES: We report a case of the allogenic BMT recipient, who developed ulcerative esophagitis which progressed to generalized HSV infection and graft versus host reaction (GVHR).We consider several diagnostic approaches to detection of active HSV infection in this patient. STUDY DESIGN: Polymerase chain reaction (PCR) was used to detect HSV DNA in esophageal biopsy specimens and peripheral leukocytes (PBL). Isolation of HSV in tissue culture was performed to prove infectious virus in swabs from mucocutaneous lesions or in PBL. RESULTS: Using PCR, HSV DNA was detected in peripheral leukocytes of the patient who had developed generalized HSV infection accompanied with hepatosplenomegaly and hepatitis. At that time, a fully infectious ACV-resistant HSV was isolated from his PBL. On the other hand, HSV DNA was not detected in PBL of other BMT-recipients with skin- or organ-localized infection. CONCLUSIONS: Presence of HSV-DNA in PBL of BMT recipients can signalize generalized HSV infection. Isolation of HSV from PBL by cocultivation with human fibroblasts can be used as an alternative diagnostic approach in these patients.

Identification of seroreactive epitopes of human papillomavirus type 18 E7 protein by synthetic peptides.

Nine everlapping peptides covering the entire sequence of early protein E7 of human papillomavirus type 18 (HPV-18) were synthesized and tested as antigens with pools of selected human sera in ELISA. Peptides denoted 18/E7-2, 18/E7-3, and 18/E7-5 (amino acid positions 11-33, 21-40, and 41-60, respectively) were reactive with pooled sera originating from HPV-18 DNA-positive cervical cancer patients but not with sera from HPV-16 DNA-positive cervical cancer patients or from condyloma acuminata patients. This suggested that the epitopes contained in these peptides were HPV-18 type-specific, relative to HPV types 16, 11, and 6. On the other hand, 18/E7-1 (aa 1-23) and 18/E7-6 (aa 51-70) peptides were cross-reactive. The prevalence of antibodies reactive with 18/E7-2, 18/E7-3, and 18/E7-5 peptides in cervical carcinoma patients was very low. Thus, the utilization of these peptides for monitoring HPV-18 infection seems to be rather limited.

Epitope mapping of the human papillomavirus type 16 E4 protein by means of synthetic peptides.

Eight overlapping icosapeptides covering the entire sequence of the E4 protein of human papillomavirus type 16 (HPV-16), were prepared and tested for their reactivity with human sera in IgG-specific ELISA. The strongest reactivity of sera from HPV-16 DNA-positive invasive cervical carcinoma (INCA) patients was detected with the peptide denoted 16/E4-6, covering amino acids 51 to 70. Subsequently nearly 200 sera were tested for the presence of the 16/E4-6-specific antibody. Reactivity was more frequent in cervical intraepithelial neoplasia patients and INCA patients than in matched control subjects. Sera from INCA patients were also tested for antibody reactive with peptide 16/E7-2 covering the major type-specific reactive region of the HPV-16 E7 protein. Only four of 13 sera possessing the 16/E4-6-specific antibody were reactive with the 16/E7-2 peptide.

Comparison of ELISA and western blotting for human papillomavirus type 16 E7 antibody determination.

A total of 140 sera originating from healthy women and women with either cervical intraepithelial neoplasia or cervical cancer were tested for the presence of IgG antibody against E7 of human papillomavirus type 16 (HPV-16) by ELISA using a synthetic icosapeptide, denoted 16/E7-2, representing amino acids 11 to 30, and by Western blotting (WB) using a genetically engineered HPV-16 E7 fusion protein. Eighteen sera were found positive in either one or the other test. Positive reactions were more frequently detected in cervical carcinoma patients (12 of 34, 35.2%) than in the other individuals (six of 106, 5.7%). Ten children's (1 to 3 years of age) sera reacted in neither ELISA nor WB with HPV-16 E7. A high degree of concordance between the two tests was found suggesting that both tests detect the same or similar activity. To locate the reacting epitopes in the E7 protein, absorption tests were performed with peptides corresponding to various sections of the protein. Based on the results obtained, sera possessing antibody to HPV-16 E7 could be differentiated into those reactive with only the 16/E7-2 peptide and those reactive with other HPV-16 E7 epitopes.

Synthetic peptides derived from E7 region of human papillomavirus type 16 used as antigens in ELISA.

Nine overlapping peptides (20 amino acid) covering the entire sequence of early antigen E7 of human papillomavirus type 16 (HPV-16) were synthesized and tested as antigens with human sera in ELISA. Five of these peptides (no. 1 to 5 counting from the N terminus of the E7 protein) reacted with a pool of sera from HPV-16-infected individuals (as determined by molecular hybridization with their biopsy specimens); one (no. 5) was also reactive with pools of HPV-18- and HPV-6- or 11-infected individuals. Sera from 24 patients with cervical intraepithelial neoplasia (CIN) and from 29 invasive cervical carcinoma (INCA) patients were tested for the presence of antibodies reactive with peptides, no. 1 to 4 covering amino acids 1 to 50 and with peptide no. 5 covering amino acids 41 to 60. Only one of the sera from CIN patients was reactive with peptides no. 1 (amino acids 1 to 20) and no. 4 (amino acids 31 to 50). However, the majority of these sera reacted with peptide no. 5. The occurrence of this antibody was only slightly less frequent in sera from healthy subjects compared to CIN patients. On the other hand, sera from the INCA patients were reactive with the peptides no. 1 to 3 more frequently than the sera from matched control subjects. Positive reactions of sera from INCA patients were most frequently seen with no. 2; 24% of these sera but only 7% of the controls were reactive with no. 2 peptide. The present data suggest that no. 1 to 3 are HPV-16-specific, whereas no. 5 is broadly cross-reactive.

Presence of antibody reactive with synthetic peptide derived from L2 open reading frame of human papillomavirus types 6b and 11 in human sera.

Nine oligopeptides corresponding to segments of different open reading frame (ORF) proteins of human papillomavirus (HPV) 6b and HPV-16 were prepared and tested for reactivity with human sera in enzyme-immunoassay (ELISA). Of these only heptadecapeptide derived from L2 ORF of HPV-6b, and encoded also by L2 ORF of HPV 11, was reactive with some human sera. Over 400 human sera of different origin were tested for the presence of antibody to this antigen. While less than 15% of sera from healthy subjects or cervical carcinoma patients were found antibody positive, sera from the majority of condylomata accuminata (CA) patients were reactive. The antibody titres varied from 1:10 (initial serum dilution) to 1:80; in this respect there was no marked difference between sera from CA patients and the other subjects. The prevalence of antibody was higher among promiscuous than nonpromiscuous women. This is in line with the assumption that sexual intercourse is the most important route of HPV 6 and 11 transmission.

A general procedure for evaluation of immunological relevance of synthetic peptides: peptides synthesized on paper in enzyme-linked immunosorbent assay.

Multiple continuous-flow solid-phase peptide synthesis has been adapted for synthesis of peptides on a cellulose carrier (Whatman 3MM paper). Paper-bound synthetic peptides that represent antigenic determinants of particular proteins detected antibodies against the respective proteins in an enzyme-linked immunosorbent assay. The method is applied to the synthesis, and use in site-directed serology, of four peptides derived from the gp41 glycoprotein of HIV, the Epstein-Barr virus-determined nuclear antigen-1 and VCA proteins of the Epstein-Barr virus, and the early region of human papillomavirus type 11.

Some immunological characteristics of subjects suffering from frequent herpes simplex virus recrudescences.

Some parameters of specific and non-specific immunity were tested in a group of 44 subjects suffering from frequent herpes simplex type 1 (HSV-1) or herpes simplex type 2 (HSV-2) recrudescences. The tests performed included determinations of (i) HSV complement-independent and complement-dependent neutralizing antibodies, (ii) antibodies to glycoprotein C of HSV-1 and glycoprotein G of HSV-2, (iii) antibodies to viral capsid and early antigens of Epstein-Barr virus, (iv) antibodies to tetanus toxoid, (v) serum levels of IgM, IgG, IgA, transferrin, prealbumin and C'3 and C'4 components of complement, (vi) active and total T lymphocytes, (vii) phagocyting activity of polymorphonuclear neutrophils, eosinophils and mononuclear cells, (viii) skin reactivity to tuberculin, toxoplasmin, candidin, tetanus and diphtheria toxoids. In the patients the following deviations from the control groups were noted: (i) Antibody levels to homotypic but not to heterotypic HSV were enhanced, (ii) serum IgM levels were elevated, (iii) percentages and numbers of active and total T lymphocytes were decreased, (iv) phagocyting activity of neutrophils was depressed but that of eosinophils was increased.

[Non-differentiated carcinoma of the nasopharynx in children]. / K problému nediferencovaného karcinomu nosohltanu u detí.

The authors deal in their paper with the problem of nondifferentiated carcinoma in childhood. They draw attention to data in the literature which report an incidence of 1-15% and compare with these data their own experience according to which in 158 patients with carcinoma of the nasopharynx there were seven children. They emphasize the inexpressive character of symptoms and thus the relatively long time which elapses before causal anti-tumourous treatment is started in child patients. They emphasize the importance of a uniform diagnostic protocol where they accentuate the directoscopy of the Rosenmüller fossa, tympanometry, computed tomography and draw attention to the advantages of EBV serology and mapping according to the HLA system. As far as treatment is concerned, they recommend radiotherapy, supplemented by adjuvant administration of the transfer factor and Mitolactol. In the conclusion the authors emphasize the necessity of team work to ensure correct interpretation of results from the diagnostic sphere and to ensure treatment in the initial stages when therapy gives the most permanent results.

Prospective study on the relationship between cervical neoplasia and herpes simplex type 2 virus. III. Presence of herpes simplex type-2 antibody in sera of subjects who developed cervical neoplasia later in the study.

Paired sera taken from 63 subjects who developed cervical neoplasia in the course of a prospective study on this disease were examined for the presence of herpes simplex virus type 2 (HSV-2) antibody. The first serum was taken at enrollment into the study, while the second was obtained after development of the disease, i.e. 2-4 years later. Simultaneously, paired sera from a group of control subjects, who remained free of any cytological and colposcopical abnormalities throughout the study, were also investigated. Controls were matched with patients by age, age at first intercourse, number of sexual partners, smoking habits and history of diathermoelectrocoagulation of ectopic epithelium and transformation zone of the cervix. The first sera from these subjects were obtained at enrollment while the second ones were taken at the end of the study, i.e. 5 to 7 years later. Antibody titres were remarkably stable in both patients and control subjects. Seroconversions from HSV-2 negativity to HSV-2 positivity as measured by the increase in the II/I ratio or development of antibody reactive with HSV-2-specific glycoprotein G were rare and no significant differences between the patients and control subjects were detected. This indicates that the development of the disease was apparently not followed by immediate or early activation of latent HSV-2 infection.

Determination of herpes simplex virus type-specific antibodies by solid-phase RIA on Helix pomatia lectin-purified antigens.

Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) antibodies in humans and animals were determined by a micro-solid-phase radioimmunoassay with Helix pomatia lectin/Sepharose 4B-purified antigens from HSV-1- and HSV-2-infected cells. A low but constant heterotypic reactivity was found with HSV-1 antigen, whereas HSV-2 antigen was type specific, as evidenced by (1) its reactivity with sera from HSV-2- but not with those from HSV-1-immunized animals, (2) its reactivity with monoclonal antibody to glycoprotein C but not with that to other HSV-2 glycoproteins, and (3) the negative results obtained in serum samples from 57 children, of whom 49 possessed HSV-1 neutralizing antibody. Antibody to HSV-2 (anti-HSV-2) was detected in serum samples from 14 of 16 subjects treated for genital herpetic lesions. The prevalence of anti-HSV-2 in women did not differ in the 25-35- and 36-45-year-old age groups but did correlate with the number of sex partners, with only 10% of women reporting one sex partner and 33% of those reporting more than 10 sex partners having anti-HSV-2.

Prospective study on the relationship between cervical neoplasia and herpes simplex type-2 virus. II. Herpes simplex type-2 antibody presence in sera taken at enrollment.

Sera obtained at enrollment in the study from patients suffering from moderate to sever dysplasia (cervical intraepithelial neoplasia grade II), carcinoma in situ (cervical intraepithelial neoplasia grade III) and invasive carcinoma, or developing any of these conditions in the course of the prospective study, and from control subjects, were examined for herpes simplex type-2 (HSV-2) antibody presence. The controls were matched with the patients by age, age at first intercourse, number of sexual partners, smoking habits and history of diathermoelectrocoagulation of the ectopic epithelium and transformation zone of cervix. Only those subjects were selected as controls who remained free of pathological colposcopical and cytological findings throughout the observation period, i.e. for at least 4 years after their serum sample was obtained. The microneutralization test (MNT) and type-2-specific solid-phase radioimmunoassay (SPRIA) were used as serological tests. No difference in the prevalence of HSV-2 antibody between the patients and controls was revealed by either test. Various combinations of the results from the two tests also failed to show any difference between patients and controls. Moreover, no significant differences were observed in the prevalence of HSV-2 antibody between patients suffering from the various pathological conditions and those diagnosed at enrollment and later in the course of the study. These results do not provide any support for the hypothesis of the involvement of HSV-2 in cervical neoplasia.

Presence of Epstein-Barr virus DNA in tonsillar tissues.

Sera from 48 tonsillar carcinoma (TC) patients, 48 matched controls and 16 recurrent exudative tonsillitis (RET) patients were examined for the presence of Epstein-Barr virus (EBV) associated nuclear antigen (EBNA), early antigen (EA) and virus capsid antigen (VCA). Higher prevalence and significantly higher antibody titres against all three EBV-associated antigens were observed in TC patients in comparison with controls and RET patients. Patients suffering from anaplastic TC had higher titres of antibodies against VCA and EA than TC patients with other histological diagnoses. Five out of 11 TC biopsies obtained from 9 patients were positive for EBV DNA at levels of 0.17, 4 to 5, 15 to 18 and in two cases 3 EBV genome equivalents per cellular genome. Among 16 RET patients, 4 were found positive at levels not exceeding 2.17 EBV genome equivalents per cellular genome. Higher titres of antibody against all EBV antigens were found in TC and RET patients with EBV DNA-positive tonsillar tissue than in those with EBV DNA-negative tonsillar tissue.

Binding of SV40 tumour antigen and Epstein-Barr-virus nuclear antigen to isolated acid-fixed nuclei.

SV40 tumour antigen (TA) bound to methanol-acetic-acid-fixed interphase nuclei and metaphase chromosomes. This reaction was visualized by the anti-complement immunofluorescence test. The reaction was negative when the nuclei had been treated with deoxyribonuclease prior to the addition of antigen, or when TA-antibody negative serum had been used. In parallel tests TA and Epstein-Barr virus nuclear antigen were examined. Both the binding patterns and the optimum conditions for the reaction were similar.