[Prevention of nosocomial transmission of human immunodeficiency virus (HIV) from HIV-positive healthcare workers. Recommendations of the German Association for the Control of Viral Diseases (DVV) e.V. and the Society for Virology (GfV) e.V]. / Prävention der nosokomialen Übertragung von humanem Immunschwächevirus (HIV) durch HIV-positive Mitarbeiterinnen und Mitarbeiter im Gesundheitswesen. Empfehlungen der Deutschen Vereinigung zur Bekämpfung der Viruskrankheiten (DVV) e.V. und der Gesellschaft für Virologie (GfV) e.V.

To the best of our knowledge, the German Association for the Control of Viral Diseases (DVV) e.V. and the Society for Virology (GfV) e.V. are the first in Europe to provide precise recommendations for the management of health care workers (HCWs) who are infected with human immunodeficiency virus (HIV). Requirements for HIV-infected HCWs need to be clearly defined. With a permanent viral burden of less than or equal to 50 copies/mL, HIV-positive HCWs are allowed to perform any surgery and any invasive procedure, as long as the infected HCW uses double-gloving, undergoes follow-up routinely by occupational medicine professionals, undergoes a quarterly examination of viral burden, and has a regular medical examination by a physician who has expertise in the management of HIV. Unrestricted professional activity is only possible with a strict compliance to take antiretroviral therapy and if the HIV-infected HCW strictly adheres to the recommended infection control procedures. Complete compliance with the recommendation almost certainly leads to no HIV transmission risk in patient care.

Targeted release of oncolytic measles virus by blood outgrowth endothelial cells in situ inhibits orthotopic gliomas.

Malignant gliomas remain largely incurable despite intensive efforts to develop novel therapies. Replicating oncolytic viruses have shown great promise, among them attenuated measles viruses of the Edmonston B strain (MV-Edm). However, host immune response and the infiltrative nature of gliomas limit their efficacy. We show that human blood outgrowth endothelial cells (BOECs), readily expandable from peripheral blood, are easily infected by MV-Edm and allow replication of MV-Edm while surviving long enough after infection to serve as vehicles for MV-Edm (BOEC/MV-Edm). After intravenous and peritumoral injection, BOEC/MV-Edm deliver the viruses selectively to irradiated orthotopic U87 gliomas in mice. At the tumor, MV-Edm produced by the BOECs infect glioma cells. Subsequent spread from tumor cell to tumor cell leads to focal infection and cytopathic effects that decrease tumor size and, in the case of peritumoral injection, prolong survival of mice. Since MV-Edm within BOECs are not readily neutralized and because BOEC/MV-Edm search and destroy glioma cells, BOEC/MV-Edm constitute a promising novel approach for glioma therapy.

CMV monitoring using blood cells and plasma: a comparison of apples with oranges?

Quantitative cytomegalovirus (CMV) monitoring is still far from being standardized between transplant centers. In the present study, we compared assays for quantitative CMV monitoring using blood cells and plasma. Four hundred and thirty-five consecutive samples from 29 patients with active CMV infection after allogeneic T-cell-depleted hemopoietic stem cell transplantation were tested in parallel using pp65 antigenemia and quantitative CMV polymerase chain reaction (PCR) in blood cells and plasma (COBAS AMPLICOR CMV MONITOR). Although only 142 (53.1%) of 253 positive samples were concordantly identified by all three assays, the number of positive samples detected by each assay was not different and the quantitative values were correlated, provided that nucleic acid (NA) in plasma was isolated by COBAS AmpliPrep and not by the manual protocol. Six (18%) of 34 episodes with active CMV infection were not detected using CMV PCR in plasma; whereas in times of white blood cell aplasia or blast crisis of leukemia, samples with active CMV infection in plasma could not be detected using blood cells. We conclude that CMV monitoring in whole blood could be favorable compared with assays using plasma or blood cells alone. Automated NA isolation could become an attractive tool for a more sensitive and better standardized molecular diagnostics.

Fatal outcome in a patient developing Epstein-Barr virus-associated lymphoproliferative disorder (EBV-LPD) without measurable disease.

A 51-year-old female patient in the first chronic phase of CML received an allogeneic PBSCT from a matched unrelated donor. The transplant was manipulated by CD34+ cell selection. On day +193 after transplantation the patient was readmitted to the hospital with recurrent fever of unknown origin and cough. Clinical, radiographic and sonographic evaluation revealed no characteristic findings besides a mild splenomegaly. Screening for EBV, CMV, RSV and HSV did not indicate an active infection. On day +203 the patient developed generalized seizures, respiratory failure and died within 24 h in multiorgan failure. The macroscopic postmortem was still not enlightening; the histological examination however, demonstrated diffuse organ infiltration by monoclonal lymphoblastoid cells due to EBV-LPD.

Functional regions of the human cytomegalovirus protein pUL97 involved in nuclear localization and phosphorylation of ganciclovir and pUL97 itself.

In order to identify functional regions of the human cytomegalovirus protein pUL97 (i) different 5' fragments of the UL97 open reading frame (ORF) were fused to the coding region of the green fluorescent protein and (ii) recombinant vaccinia viruses (rVV) were generated carrying two full-length and 11 mutated UL97 ORFs. The results indicated the presence of an N-terminal region within pUL97 which changed the intracellular distribution of the fusion proteins. pUL97 was localized in the nucleus, but not in the nucleoli, and was detected in the nuclear matrix fraction. Expression of all pUL97 mutants could be confirmed by Western blot analysis. pUL97-associated ganciclovir (GCV) phosphorylation in rVV-infected cells, determined quantitatively by HPLC analysis, was abolished completely using individual UL97 deletion mutants. Phosphorylation of full-length and some of the mutated pUL97 was detected in cells infected with the rVVs. The UL97 constructs carrying point mutations from GCV-resistant HCMV isolates at positions 460M, 520H, 594V, and the 4 aa deletion 590AACR593, also resulted in decreased but not abolished phosphorylation of GCV in the rVV system, whereas the phosphorylation of pUL97 itself was not influenced. The rVV system is a suitable method for quantitatively testing the functional relevance of pUL97 mutations.

HCV and HGV in B-cell non-Hodgkin's lymphoma.

BACKGROUND/AIMS: A causative role of hepatitis C virus infection (HCV) has been discussed in the pathogenesis of mixed cryoglobulinaemia and in B-cell non-Hodgkin's lymphoma. No data are available concerning the newly discovered hepatitis G virus (HGV) and extrahepatic manifestations such as haematological malignancies. But, HCV and HGV most probably belong to the same family of Flavivirus. Consequently, we looked for the prevalence of HCV, HGV and cryoglobulins in patients with B-cell non-Hodgkin's lymphoma. METHODS: Serum samples from 69 patients with non-Hodgkin's lymphoma were studied. Diagnosis of non-Hodgkin's lymphoma was established according to the Kiel classification. Active HCV- and HGV infections were investigated using polymerase chain reaction for detection of viral RNA. Cryoglobulins were detected from serum and monoclonal immunoglobulin components were analysed with immunofixation electrophoresis. In addition, we assessed the clinical course of HCV- and HGV-infected patients under chemotherapy. RESULTS: Three of 69 (4.3%) patients with B-cell non-Hodgkin's lymphoma were HCV-infected and nine non-Hodgkin's lymphoma patients (13.0%) were positive for hepatitis G virus RNA. All HGV infected patients were suffering from low-grade non-Hodgkin's lymphoma. No HGV-infected patient was co-infected by HCV and neither HCV- nor HGV-infected patients showed clinical signs of chronic liver disease before, during or after chemotherapy. Serum samples from all patients were devoid of cryoglobulins. CONCLUSIONS: HCV seems to have no significance for the pathogenesis of non-Hodgkin's lymphoma in Germany. The increased prevalence of hepatitis G (16.3%) in patients with low-grade non-Hodgkin's lymphoma could suggest a pathological consequence of HGV infection outside of the liver. Evidence of clinically relevant hepatic disease in HGV infected patients was not obtained. Further, chemotherapy does not seem to affect the subsequent clinical course of HGV infection.

Sexually transmitted diseases in Africa: time for action.

PIP: Sexually transmitted diseases (STDs) are major public health problems which often lead to serious complications and sequelae, including infertility. Infection with STDs also facilitates the transmission of HIV, making the early diagnosis and care of STDs integrated into other services one of the most cost-effective strategies to prevent the spread of HIV. The direct and indirect costs of STDs worldwide are considerable. Sub-Saharan Africa ranks first in STD yearly incidence compared to other world regions. The World Health Organization has estimated that every year in Africa there are 3.5 million cases of syphilis, 15 million cases of chlamydial disease, 16 million cases of gonorrhea, and 30 million cases of trichomoniasis. STDs are a high public health priority especially because of their widespread prevalence and treatability. Herpes simplex virus infection and human papillomavirus infection are growing problems in sub-Saharan Africa. While STDs are caused by more than 20 microorganisms, they present themselves mainly in 4 syndromes and may therefore be treated syndromically. Africa must implement effective and comprehensive integrated activities against the STD epidemic. Elements of such a strategy will include disease prevention, screening and case finding, and the early diagnosis and treatment of cases.^ieng

Hacia donde se encamina la epidemia de infección por VIH y SIDA? / HIV and AIDS: where is the epidemic going?

Routine surveillance of HIV (human immunodeficiency virus) infection and AIDS has been established over the past decade in many countries around the world. HIV estimates derived from empirical data are essential to the assessment of the HIV situation in different parts of the world and trends are used in tracking the development of regional epidemics, thereby keeping intervention activities focused on realities. As of the end of 1995, and following an extensive country-by-country review of HIV/AIDS data, a cumulative total of 6 million AIDS cases were estimated to have occurred in adults and children worldwide and currently 20.1 million adults are estimated to be alive and infected with HIV or have AIDS. Of the total prevalent HIV infections, the majority remain concentrated in eastern, central and southern Africa, but the epidemic is evolving with spread of infection from urban to rural areas, as well as to West and South Africa, India and Southeast Asia, and to a lesser extent­with proportional shifts to heterosexual infections­in North America, Western Europe and Latin America. While the longer-term dimensions of the HIV epidemic at global level cannnot be forecast with confidence, WHO currently projects a cumulative total of close to 40 million HIV infections in men, women and children by the year 2000. By that time, the male:female ratio of new infections will be close to 1:1. Recent trends indicate that HIV prevalence levels may be stabilizing or even decreasing among pregnant women in southern Zaire and parts of Uganda, among military recruits aged 21 in Thailand, and in some populations of northern Europe and the USA. While these changes may take place as part of the intrinsic dynamic of the epidemic, there is some evidence that declines in HIV prevalence are related to declines in HIV incidence which are, at least partly, due to prevention efforts. The challenge of surveillance and evaluation methods is now to identify the ingredients of success which may reveal a glimmer of hope.

En el transcurso del último decenio, muchos países en todo el mundo han establecido una vigilancia sistemática para casos de sida y de infección por virus de la inmunodeficiencia humana (VIH). Las estimaciones de la carga de infección por VIH que derivan de datos empíricos son indispensables para evaluar la situación en diferentes partes del mundo y las tendencias se emplean para seguir la trayectoria de epidemias regionales y así poder basar las actividades de intervención en hechos concretos. Se estimó a fines de 1995, a raíz de un extenso análisis de datos sobre la infección por VIH y el sida efectuado país por país, que ha habido un total acumulativo de 6 millones de casos de sida en adultos y niños en todo el mundo y que actualmente 20,1 millones de adultos están infectados por VIH o tienen sida. De la prevalencia total de infecciones por VIH, la mayor parte se concentra en África oriental, central y meridional, pero la epidemia está evolucionando y la infección se está propagando de las zonas urbanas a las rurales, y también al sur y occidente de África, a la India y al Asia Sudoriental y, en menor escala ­con cambios similares en la proporción de infecciones heterosexuales­, a América del Norte, Europa occidental y América Latina. Aunque no se puede confiablemente pronosticar en el largo plazo la magnitud de la epidemia de infección por VIH en el mundo, la proyección actual de la OMS es de un total acumulativo cercano a los 40 millones de infecciones por VIH en hombres, mujeres y niños para el año 2000. Llegado ese año, la razón de infecciones en hombres a infecciones en mujeres se aproximará a 1:1. Las tendencias más recientes indican que las tasas de prevalencia de VIH podrían estar en vías de estabilización o incluso de reducción en mujeres embarazadas en el sur del Zaire y en partes de Uganda, en reclutas militares de 21 años de edad en Tailandia y en algunas poblaciones del norte de Europa y de los Estados Unidos de América. Si bien estos cambios pueden ocurrir como parte de la dinámica intrínseca de la epidemia, hay indicios de que las reducciones de la prevalencia de la infección por VIH están relacionadas con disminuciones de la incidencia, que se deben, al menos en parte, a las actividades de prevención. El actual reto de los métodos de vigilancia y evaluación radica en descubrir aquellos ingredientes que han llevado al éxito y que pueden ofrecer un rayo de esperanza.

Flow cytometric analysis of herpes simplex virus type 1 susceptibility to acyclovir, ganciclovir, and foscarnet.

We established a quantitative flow cytometric method for determination of herpes simplex virus type 1 (HSV-1) susceptibility to acyclovir (ACV), ganciclovir, and foscarnet in vitro. Susceptibility was defined in terms of the drug concentration which reduced the number of cells expressing HSV-1 glycoprotein C (gpC) with a fluorescence intensity of > or =10(2) by 50% (IC50). Flow cytometry allowed us to use a high (1.0) as well as a low (0.005) multiplicity of infection, and determination of the IC50 was possible after one or more viral replicative cycles. IC50s were dependent on virus input and on time postinfection. In mixture experiments, 1 to 2% resistant viruses added to a sensitive strain could be detected. The results obtained by flow cytometry showed a good qualitative correlation with those achieved by cytopathic effect inhibitory assay. However, flow cytometry might detect more quantitative differences in drug susceptibility, especially among resistant strains, as confirmed also by determination of intracellular drug phosphorylation. The mean IC50s for ACV-sensitive strains were 0.45 to 1.47 microM, and those for ACV-resistant strains were between 140 and 3,134 microM. Flow cytometric analysis was fast and accurate, automatizable, and highly reproducible. Flow cytometry may be a more powerful tool than standard cytopathic effect-based assays and could have advantages for the detection of low levels of drug resistance or mixtures of sensitive and resistant virus strains.

Reactivated fulminant hepatitis B virus replication after bone marrow transplantation: clinical course and possible treatment with ganciclovir.

A female chronic hepatitis B virus carrier (HBV-DNA negative) suffered from simultaneous hepatitis B virus and cytomegalovirus reactivation after in vivo T cell depletion preceding transplantation of an in vitro T cell depleted marrow graft for treatment of acute leukaemia. Interstitial pneumonia developing after bone marrow transplantation was successfully treated with ganciclovir (day 13 until day 46). The initially unnoticed extensive hepatitis B virus replication finally led to clinical hepatitis (day 85) and liver failure (day 96). Liver transplantation was performed, but the patient died from septicaemia. Retrospective analysis of hepatitis B virus DNA revealed that the HBV replication started immediately after T cell depletion and was completely suppressed during ganciclovir administration. Screening for HBV-DNA seems to be mandatory in comparable cases, and antiviral chemotherapy should be seriously considered.

DNA vector constructs that prime hepatitis B surface antigen-specific cytotoxic T lymphocyte and antibody responses in mice after intramuscular injection.

We tested the efficiency of induction of immune responses to the small hepatitis B surface antigen (HBsAg) in mice by intramuscular DNA immunization using different vector constructs that allow high levels of HBsAg expression in mouse cells. The HBsAg-specific responses of class I-restricted cytotoxic T lymphocytes (CTL) and of B cells (serum antibody titers) were measured. Following the intramuscular inoculation of 'naked' DNA, five different vector constructs of 4-8 kb, that contained or did not contain an intron and/or the neo gene, in which HBsAg expression was driven by promoter sequences derived from the immediate early region of HCMV, the SV40 enhancer/promoter region, or a retroviral 3' LTR efficiently primed responses of class I-restricted CD8+ CTL precursors. In contrast, the constructs in which HBsAg expression was driven by HCMV-derived promoter sequences stimulated significantly higher levels of HBsAg-specific serum antibody titers after intramuscular DNA injection than the SV40 or MPSV vector constructs. Large (15 kb) episomal vector constructs did not stimulate CTL or antibody responses. The data demonstrate that: (i) intramuscular DNA immunization represents an efficient technique for priming CTL and antibody responses to HBsAg; (ii) many vectors can be constructed that express an immunogenic product after intramuscular inoculation of 'naked' DNA; (iii) the efficiency of the tested vector constructs to prime after DNA immunization, either a CTL response, or an antibody response, differs.

Formation of differentiated tissues in vivo by periodontal cell populations cultured in vitro.

The periodontium contains heterogeneous mesenchymal cell populations with various differentiation potentials. The capacity of these cells for tissue formation as well as the origin of their precursors are still not entirely defined. In this study, cells originating from different periodontal tissues were cultured in vitro, and tissue formation in vivo following orthotopic re-implantation was investigated. Cells were recovered from the alveolar bone and periodontal ligament tissue of six minipigs, and cultured cells were then grown on extracted dental roots from the homologous animals by means of co-culture in vitro. Each minipig received 2 roots covered with alveolar bone cells, 2 roots covered with periodontal ligament cells, and 2 control roots (without cells) implanted into palatal bone defects. Intravital fluorochrome labeling was performed, and two minipigs were histologically examined after 2, 4, and 12 weeks in each case. Controls showed widespread resorption and ankylosis, whereas roots covered with cultured periodontal cells exhibited tissue formation in vivo. Alveolar bone cells synthesized a calcified cellular tissue resembling cellular cementum, suggesting that cells within this population might differentiate into cementoblasts when reimplanted with a dental substrate in vivo. Periodontal ligament cells exhibited no calcified tissue formation in vivo, but cells synthesized a connective tissue with orientated fiber bundles attached to both host bone and root, resembling periodontal ligament.

In vivo/ex vivo T cell depletion for GVHD prophylaxis influences onset and course of active cytomegalovirus infection and disease after BMT.

Combined in vivo/ex vivo T cell depletion is effective in the prophylaxis of graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT), but influences the occurrence of active cytomegalovirus (CMV) infection and disease. Twenty nine patients receiving a T cell-depleted marrow graft (Campath-1M) after intravenous application of the monoclonal antibody Campath-1G prior to conditioning were monitored for virus shedding and antigenaemia from day -7 to day +100. In seropositive patients in this group active CMV infection occurred more frequently (10 of 16) and much earlier (nine of 10 until day +21) than in 27 seropositive patients (10 of 27, P < 0.02) receiving cyclosporin A and methotrexate (CsA/MTX). Early active CMV infection after in vivo/ex vivo T cell depletion correlated strictly with an early increase in blood lymphocyte counts (P < 0.01), with predominance of NK cells and/or CD8+ T cells. Three cases of very early interstitial pneumonitis (IP) occurred after in vivo/ex vivo T cell depletion compared with none in the CsA/MTX group. IP was fatal in the only patient with early active CMV infection, who remained lymphocytopenic till death on day +31. This may indicate that an early immune response against CMV is possible and essential for favourable clinical outcome.

Male circumcision, sexually transmitted disease, and risk of HIV.

Our objective was to describe associations among male circumcision, behavioral and demographic variables, ulcerative and nonulcerative sexually transmitted disease (STD), and human immunodeficiency virus (HIV) infection via a cross-sectional study in Kigali, the capital of Rwanda. Our subjects were 837 married men who volunteered for HIV testing and counselling. Uncircumcised men had a relatively low-risk profile in that they reported fewer lifetime sexual partners and prostitute contacts than circumcised men and were more likely to live in rural areas with lower HIV prevalence rates. Uncircumcised men were also less likely to report a history of sexually transmitted disease (64% versus 73%, p = 0.01), although they were more likely to report genital ulceration (GUD) (24% versus 17%, p < 0.03) and to have inguinal adenopathy noted on physical exam (42% versus 29%, p = 0.009). Despite the low-risk profile, uncircumcised men had a higher prevalence of HIV infection than circumcised men (29% versus 21% HIV positive, p = 0.02), which was most marked in men reporting five or more lifetime sex partners (36% versus 23% HIV positive, p = 0.005) or contact with prostitutes (35% versus 23% HIV positive, p = 0.009). Circumcision remained a predictor of HIV infection in multivariate analyses (multivariate odds ratio 1.69, 95% confidence interval 1.16-2.47). Lack of circumcision is associated with a higher risk of HIV infection in Rwandan men. Further research is needed to determine whether this higher risk is due in part to poor hygiene or to complex mechanisms operating through the acquisition of other sexually transmitted diseases. Circumcision may be an appropriate risk reduction approach for men with known exposures to the virus when there are constraints to alternatives, such as condom use.

Human cytomegalovirus UL97 kinase confers ganciclovir susceptibility to recombinant vaccinia virus.

We analyzed whether the phosphotransferase encoded by the UL97 open reading frame of human cytomegalovirus (HCMV) alone is sufficient to confer ganciclovir (GCV) susceptibility to a foreign virus. Two vaccinia virus recombinants (T1 and A5) containing the UL97 open reading frames from a GCV-sensitive HCMV and from a GCV-resistant strain were constructed. T1 exhibited a GCV-sensitive phenotype in plaque reduction assays, whereas A5 did not. Moreover, T1-infected cell cultures showed a strongly increased incorporation of [14C]GCV triphosphate into macromolecular DNA, compared with recombinant A5 or vaccinia virus controls, which could be inhibited by the addition of guanosine. This shows that UL97 kinase is the only additional gene product required to make vaccinia virus susceptible to GCV, and guanosine seems to be one natural substrate for the enzyme. The system described here should be very helpful for fast and detailed functional analyses of UL97 mutations found in GCV-resistant HCMV isolates.

Prevention indicators for evaluating the progress of national AIDS programmes.

PIP: Major interventions to reduce HIV transmission involve increasing knowledge about preventing HIV transmission for sustained behavioral changes; and enhancing the control of sexually transmitted diseases (STD), which increase the probability of HIV transmission. Activities have also been developed to prevent the transmission of HIV by blood, donor selection, and more rational use of transfusions. Behavioral changes among injecting drug users have also been promoted. Recommendations are made for the evaluation of AIDS programs, focusing on prevention of sexual transmission of HIV, and outlining the approach developed by the Global Program on AIDS (GPA; Geneva, Switzerland) for use by national programs. Based on the feasibility, accuracy, reliability and validity of the quantitative assessment of programs, 10 indicators of progress and outcomes of prevention activities have been developed by GPA. These include indicators of population knowledge regarding preventive practices, reported sexual behavior and use of condoms in the general population, STD service evaluation, and indicators of program impact. The latter are measured through the reported STD incidence in the general male population, and syphilis and HIV prevalence in women. The four methods are proposed for measuring the 10 core prevention indicators (PI). Five PIs are measured during a population survey: reported knowledge of preventive practices (PI-1), condom availability at peripheral level (PI-3), reported frequency of nonregular sexual partners (PI-4), reported condom use during nonregular sexual encounters (PI-5), and reported STD incidence among men (PI-9). Condom availability at central level (PI-2) is assessed through key-informant interviews with major distributors. Structured health facility surveys allow assessment of the appropriateness of STD case management (PI-6 and PI-7). A serosurvey among antenatal clinic attenders aged 15-24 years allows the measurement of HIV and syphilis seroprevalence in that population (PI-8 and PI-10). GPA recommends that such surveys be repeated after a period of 1 to several years.^ieng