Severe underquantification of HIV-1 group O isolates by major commercial PCR-based assays.

HIV-1 diversity poses major challenges to viral load assays because genetic polymorphisms can impede nucleic acid detection. In addition to the on-going viral diversification within the HIV-1 group M pandemic, HIV-1 genetic diversity is further increased by non-group M infections, such as HIV-1 groups O (HIV-1-O), N and P. We here conducted a systematic evaluation of commercially available PCR assays to detect HIV-1-O isolates. We collected 25 primary HIV-1-O isolates covering all genetic clusters within HIV-1-O. Subsequently, this panel of isolates was tested on eight commercially available quantitative and five qualitative HIV-1 PCR-based assays in serial dilutions. Sequence analyses were performed for severe cases of underquantification or lack of detection. We observed differences between the assays in quantification that depended on the HIV-1-O isolate's subgroup. All three tested HIV-1-O subgroup IV isolates were underquantified by the Roche CAP/CTM >800-fold compared to the Abbott RealTime assay. In contrast, the latter assay underquantified several subgroup I isolates >200-fold. Notably, the Xpert HIV-1 Viral Load test from Cepheid failed to detect two of the HIV-1-O isolates, whereas the Roche Cobas 8800 assay readily detected all isolates. Comparative sequence analyses identified polymorphisms in the HIV-1-O long-terminal repeat and integrase genes that likely underlie inadequate nucleic acid amplification. Potential viral load underquantification should be considered in therapeutic monitoring of HIV-1-O-infected patients. Pre-clinical assessments of HIV-1 diagnostic assays could be harmonized by establishing improved and internationally standardized panels of HIV-1 isolates that cover the dynamic diversity of circulating HIV-1 strains.

[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.

[Emerging viral diseases in Europe]. / "Neue" Viren als bedrohliche Krankheitserreger in Europa.

Emergence of viral agents in Europe is influenced by various factors. Climatic changes influencing possible vectors, insufficient vaccination, and travel of man and goods are among the most important reasons to explain these changes. Fever and arthralgia are the leading symptoms in infection with Dengue, Sindbis, or Chikungunya virus. In contrast, tick-born encephalitis (TBE), Toscana, or West Nile virus infections mainly lead to meningo-encephalitis. In Europe, hemorrhagic fever is caused by Crimean Congo and Hanta virus. Protective vaccines are available for emerging viral agents like TBE, influenza and measles.

Mutations in the C-terminal region of the HIV-1 reverse transcriptase and their correlation with drug resistance associated mutations and antiviral treatment.

OBJECTIVE: replication of HIV-1 after cell entry is essentially dependent on the reverse transcriptase (RT). Antiretroviral drugs impairing the function of the RT currently aim at the polymerase subunit. One reason for failure of antiretroviral treatment is the evolvement of resistance-associated mutations in the viral genome. For RT inhibitors, almost all identified mutations are located within the polymerase; therefore, general genotyping confines to investigate this subunit. Recently several studies have shown that substitutions within the RNase H and the connection domain increase antiviral drug-resistance in vitro, and some of them are present in patient isolates. AIM: the aim of the present study was to investigate the prevalence of these substitutions and their association with mutations in the polymerase domain arising during antiretroviral treatment. MATERIAL AND METHODS: we performed genotypic analyzes on seventy-four virus isolates derived from treated and untreated patients, followed at the HIV Centre of the Johann Wolfgang Goethe University Hospital (Frankfurt/Main, Germany). We subsequently ana?lysed the different substitutions in the c-terminal region to evaluate whether there were associations with each other, n-terminal substitutions or with antiretroviral treatment. RESULTS: We identified several primer grip substitutions, but almost all of them were located in the connection domain. This is consistent with other in-vivo studies, in which especially the primer grip residues located in the RNase H were unvaried. Furthermore, we identified other substitutions in the connection domain and in the RNase H. Especially E399D seemed to be associated with an antiretroviral treatment and N-terminal resistance-delivering mutations. CONCLUSION: some of the identified substitutions were associated with antiviral treatment and drug resistance-associated mutations. Due to the low prevalence of C-terminal mutations and as only a few of them could be associated with antiviral treatment and N-terminal resistance-delivering mutations, we would not recommend routinely testing of the C-terminal RT region.

Human immunodeficiency virus type-1 group M quasispecies evolution: diversity and divergence in patients co-infected with active tuberculosis.

The evolution of intra-host human immunodeficiency virus type 1 (HIV-1) quasispecies prior and after treating active tuberculosis (TB) with chemotherapy in HIV-1/TB patients was assessed. Two time points HIV-1 quasispecies were evaluated by comparing HIV-1-infected patients with active tuberculosis (HIV-1/TB) and HIV-1-infected patients without tuberculosis (HIV-1/non-TB). Plasma samples were obtained from the Frankfurt HIV cohort, and HIV-1 RNA was isolated. C2V5 env was amplified by PCR and molecular cloning was performed. Eight to twenty-five clones were sequenced from each patient. Various phylogenetic analyses were performed. We found a significant increase in diversity and divergence in HIV-1/TB compared to the HIV-1/non-TB. For HIV-1/TB, the average rate of evolution of C2V5 env was higher than previous reports (2.4 × 10(-4) substitution/site/day). Two groups of HIV-1/TB were observed based on the rate of HIV-1 evolution and coreceptor usage: A fast evolving R5-tropic dominating group and a relatively slowly evolving X4 group. The results demonstrated that active TB has an impact on HIV-1 viral diversity and divergence over time. The influence of active TB on longitudinal evolution of HIV-1 may be predominant for R5 viruses.

Tissue donation and virus safety: more nucleic acid amplification testing is needed.

In tissue and organ transplantation, it is of great importance to avoid the transmission of blood-borne viruses to the recipient. While serologic testing for anti-human immunodeficiency virus (HIV)-1 and -2, anti-hepatitis C virus (HCV), hepatitis B surface antigen (HBsAg), anti-hepatitis B core antigen (HBc), and Treponema pallidum infection is mandatory, there is until now in most countries no explicit demand for nucleic acid amplification testing (NAT) to detect HIV, hepatitis B virus (HBV), and HCV infection. After a review of reports in the literature on viral transmission events, tissue-specific issues, and manufacturing and inactivation procedures, we evaluated the significance of HIV, HCV, and HBV detection using NAT in donors of various types of tissues and compared our results with the experiences of blood banking organizations. There is a significant risk of HIV, HCV, and HBV transmission by musculoskeletal tissues because of their high blood content and the high donor-recipient ratio. If no effective virus inactivation procedure for musculoskeletal tissue is applied, donors should be screened using NAT for HIV, HCV, and HBV. Serologically screened cardiovascular tissue carries a very low risk of HIV, HCV, or HBV transmission. Nevertheless, because effective virus inactivation is impossible (retention of tissue morphology) and the donor-recipient ratio may be as high as 1:10, we concluded that NAT should be performed for HIV, HCV, and HBV as an additional safety measure. Although cornea allografts carry the lowest risk of transmitting HIV, HCV, and HBV owing to corneal physiology, morphology, and the epidemiology of corneal diseases, NAT for HCV should still be performed. If the NAT screening of a donor for HIV, HCV, and HBV is negative, quarantine storage of the donor tissue seems dispensable. In view of numerous synergistic effects with transfusion medicine, it would be advantageous for tissue banks to cooperate with blood bank laboratories in performing virological tests.

A neutralization assay for HIV-2 based on measurement of provirus integration by duplex real-time PCR.

Specific, effective and rapid neutralization assays are crucial for the development of an HIV vaccine based on the stimulation of neutralizing antibodies and the development of such an assay for the human immunodeficiency virus-2 (HIV-2) is described. Virus neutralization was measured as the reduction of provirus integration using a duplex real-time PCR with high efficiency (99.4%). This PCR uses primers and a probe specific for the proviral LTR. Amplification and quantitative analysis of the cellular GAPDH gene was carried out in parallel to control for toxic or growth-inhibitory components in the sera. The neutralization assay was used to screen sera from 23 HIV-2 infected patients. 21 sera were able to neutralize HIV-2(60415K), 20 sera neutralized HIV-2(7312A) and 7 sera cross-neutralized HIV-1 IIIB. In contrast, when 14 of these sera were tested in parallel with a conventional neutralization assay based on a p27Gag capture ELISA, only one was found to neutralize HIV-2(60415K) and 11 to neutralize HIV-2(7312A) compared with 12 and 13 sera respectively using the PCR-based assay.

[Global warming: trailblazer for tropical infections in Germany?]. / Globale Erwärmung: Wegbereiter für tropische Infektionskrankheiten in Deutschland?

Since 1850, the CO (2) content of the atmosphere has increased from 280 to 360 ppm, and the average surface temperature has risen from 14.6 to 15.3 C . A further increase between 1.8 and 4.0 C is expected for the 21st century. Temperate and cold climate zones are affected predominantly, but tropical regions are not spared. At the same time, the world wide climate effects of the "El Niño Southern Oscillation" are amplified. Global warming enhances the growth of tropical pathogens (malarial plasmodia, leishmania, yellow fever virus, dengue virus, West Nile virus, Vibrio cholerae) and vectors (anopheles, aedes, culex, and phlebotomus mosquitos; hard ticks). Global warming may lead to the emergence of diseases which at present are not endemic in Germany, like West Nile fever, Dengue fever, or Leishmaniases, and to enhanced transmission of borreliosis and tick-borne encephalitis. Malaria and cholera, in contrast, are influenced more strongly by socioeconomic factors. Improved surveillance and intensified research on the relationship between climate change and infectious diseases is needed.

No influence of atopic diseases on antibody titres following tetanus, diphtheria and hepatitis B immunisation among adults.

Several studies have reported associations between reduced humoral immune response to vaccine antigens and diseases with modified reactions of the immune system. We have investigated the influence of atopic diseases on specific IgG levels to tetanus, diphtheria and hepatitis B (HB), following immunisation, in a general adult population. From the Study of Health in Pomerania, a total number of 3,920 subjects aged 20 to 79 years were included in the analyses. Information on immunisation history, as well as behavioural and socio-demographic characteristics were collected. Anti-tetanus IgG, anti-diphtheria IgG and anti-HBs IgG were measured by indirect enzyme-linked immunosorbent assay (ELISA). Odds ratios and 95% confidence intervals were calculated using logistic regression. Atopic diseases were reported by 14% of participants. Proportions of 67%, 34% and 10% had been vaccinated against tetanus, diphtheria and hepatitis B within the past ten years, respectively. Multi-variable analyses revealed no associations between the presence of atopic diseases and all of the three vaccine-specific antibody titres. We conclude that there is no reduced immune response related to antibody production following immunisations against tetanus, diphtheria and hepatitis B in adults with atopic diseases.

[Bilateral retinal vasculitis with arterial aneurysms]. / Bilaterálna retinálna vaskulitída s arteriálnymi aneuryzmami.

The authors reported unusual and rare condition of bilateral retinal vasculitis primarily affecting the central retinal artery at the nerve head and its 4 main branches. The most striking feature was the presence of the diffuse vitreous cells, occlusion of branch retinal artery, segmental periarterial infiltration, arterial sheating, retinal arterial aneurysms, disc swelling, peripheral retinal non perfusion and their complications. During 13 year's observation and treatment one eye went blind 3 years after initial examination. Second eye started the same clinical course two years after beginning of the disease. To avoid similar devastating course of the disease we started systemic steroids and immunosuppressive therapy, followed by photocoagulation of nonperfused peripheral retina and vitreoretinal surgery. We achieved stabilization of the disease with decreased visual functions. Comprehensive systemic work-up was unrevealing, no clear etiology was identified and diagnosis of idiopathic retinal vasculitis was made.

Enterovirus RNA sequences in sera of schoolchildren in the general population and their association with type 1-diabetes-associated autoantibodies.

Type 1 diabetes (T1D) is an autoimmune disease linked with genetic factors as well as with environmental triggers, such as virus infections, but the aetiology is still unclear. The authors analysed serum from autoantibody-positive (n=50) and autoantibody-negative (n=50) schoolchildren as well as children newly diagnosed with T1D (n=47; time from diagnosis, median 5 days, interquartile range 1-12 days) for the presence and frequency of enterovirus (EV) and adenovirus sequences. The autoantibody-positive and -negative groups were part of the Karlsburg Type 1 Diabetes Risk Study of a Normal Schoolchild Population, which represents a general population without T1D first-degree relatives. There was no significant seasonality of sampling in any of the three groups investigated. EV RNA sequences were detected in 10 of 50 (20%) autoantibody-positive children and in 17 of 47 (36%) children newly diagnosed with T1D, but only in two of 50 (4%) of the age- and sex-matched controls (P<0.05, P<0.001). Characterization of the EV amplicons by direct sequencing revealed high homology with coxsackievirus B group. For adenovirus we found no data to support an association with T1D. The data support the hypothesis that different enteroviruses may be aetiologically important as a trigger and/or accelerating factor in the process of T1D development.

[Microbiologic diagnosis of selected skin infections]. / Mikrobiologische Diagnostik ausgewählter bakterieller Hautinfektionen.

Using as examples either common or important but less frequent bacterial skin diseases, detailed practice-oriented information is provided on microbiologic diagnostic procedures. Despite the availability of many advanced techniques, often the simplest measures can provide the correct diagnosis and help guide therapy. The practicing physician must also know what is required for more advanced diagnostic procedures; if they are used improperly, unnecessary costs accumulate. Conversely, the decision process and technical methods must be understandable for those without special background in microbiology. The value of all methods is discussed from the perspective of dermatology with emphasis on appropriate sampling and handling of specimens.

Chlamydophila pneumoniae respiratory tract infection aggravates therapy refractory bronchitis or pneumonia in childhood.

BACKGROUND: Chlamydophila pneumoniae was frequently found in bronchial secretions of children with therapy-refractory bronchitis or pneumonia. It was studied, how the agent modifies the course of disease and what findings are associated with the infection. PATIENTS AND METHODS: Bronchial secretions obtained at bronchoscopy of 428 children were studied for C. pneumoniae infection using polymerase chain reaction with enzyme immunoassay detection. Children tested negative and positive were compared for their clinical findings. RESULTS: C. pneumoniae was found in 143 children (33 %). A C. pneumoniae infection has been found to be associated with a purulent bronchial inflammation (90/143 vs. 144/285, p = 0.02), a Streptococcus pneumoniae co-infection (13/143 vs. 6/285, p = 0.002) and a restrictive disturbance (11/51 vs. 8/93, p = 0.04). Purulent inflammation (Odds ratio 7.9; 95 % confidence interval [CI] 1.6-39.3), 2 co-infections (Odds ratio 14.3; 95 % CI 1.4-144.4) and co-infection with M. pneumoniae (4/4 versus 9/26, p = 0.03; Mantel Haentzel 3.0; 95 % CI 1.1-8.0) were identified as factors more often associated with a restrictive disturbance in children with bronchial C. pneumoniae infection. An adequate antibiotic therapy improved pulmonary function. No association was found for wheezing, eosinophil inflammation of the nasal mucosa, alpha-1 antitrypsin or immunoglobulin deficiency in serum, level of secretory IgA in bronchial mucus, pathological lung scintigram, gastro-esophageal reflux disease, sweat test and other co-infections. CONCLUSIONS: In children with therapy-refractory bronchitis or pneumonia bronchial C. pneumoniae infection was associated with a more severe disease in case of several, mostly bacterial co-infections. Adequate antibiotic therapy for C. pneumoniae infection has been demonstrated to improve pulmonary function.

Molecular and clinical characteristics of respiratory syncytial virus infections in hospitalized children.

The objective of this study was to determine the importance of respiratory syncytial virus (RSV) for hospitalization in the north east of Germany and to obtain molecular epidemiological data of the circulating strains. Using a rapid and sensitive reverse transcriptase-PCR, it was found that a quarter of pediatric respiratory disease admissions were due to RSV. Infections caused by RSV in hospitalized patients were determined over the whole year. Both RSV groups A and B were identified with a predominance of RSV A (86%) over the entire period. The analysis of the deduced amino acid sequences by direct sequencing showed that very similar RSV strains are circulating in the community.

[SIV as a source of HIV. On the origin of human immunodeficiency viruses from non-human primates]. / SIV als Quelle von HIVZum Ursprung der humanen Immunschwächeviren aus nicht-humanen Primaten.

It is assumed that HIV, the human immunodeficiency virus, started its spread after the Second World War. Molecular analysis of the genome of various HIV-1 types has shown that this virus can be divided into the groups M, N, and O and that these genome sequences fit perfectly to the genomes found in SIV of chimpanzees (SIVcpz) living in the area of West and Central Africa. SIVcpz is nonpathogenic for chimpanzees indicating that the virus and host have adapted for a long period. HIV-2 genome sequences converge with SIV sequences of sooty mangabey monkeys from West Africa (SIVsm), covering the subtypes A to G from HIV-2. SIVsm is nonpathogenic for mangabey monkeys. All available data indicate that HIV-1 and HIV-2 have been introduced into humans at least several times. Since SIVcpz and SIVs from other monkeys are recombinant viruses, it cannot be excluded that a new recombinant SIV might again enter the human population and initiate a new epidemic.

Are enterovirus infections a co-factor in sudden hearing loss?

Since viral infections are believed to be one of the causes of sudden hearing loss we have used serological assays for herpes simplex virus (HSV), varicella zoster virus (VZV), and enterovirus as well as polymerase chain reaction for enterovirus to test 55 sudden hearing loss patients for viral infections. Serological screening of these patients for HSV and VZV failed to reveal significant differences between the patient group and the controls. In contrast, enterovirus sequences were detected by RT-PCR in 40% of the patient group, but in none of the controls, suggesting that enterovirus infections may be associated with sudden hearing loss.

Real-time PCR to improve the diagnosis of respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is one of the most important virus respiratory pathogens in infants and young children. A rapid and sensitive diagnosis is essential to focus any outbreak due to this virus. A real-time RT-PCR method was designed using a primer/probe pair from the F gene. Simultaneously with nested RT-PCR and antigen ELISA, 71 consecutive specimens from hospitalized children with clinical symptoms of acute respiratory distress were evaluated to confirm the incidence of RSV infection. RSV was detected in 25 (35.2 %) specimens by real-time RT-PCR and in 19 (26.7 %) by nested RT-PCR. The assay was specific for RSV. The procedure offers a rapid and sensitive alternative to conventional RT-PCR. Closed-tube detection eliminates the risk of contamination.