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.

BackgroundHuman herpes virus 8 (HHV‐8), a gamma herpes virus, is the etiological agent for Kaposi sarcoma (KS). HIV‐infected adults with advanced immunodeficiency are at risk. Prevalence data of HHV‐8 infection in HIV‐infected children living in non‐endemic areas are limited. Serologic studies indicate low seroprevalence rates of 3–4% for healthy children living in United States and Germany [1].Purpose of the studyThe aim of the study was to determine the seroprevalence of HHV‐8 antibodies among vertically HIV‐infected pediatric patients in Germany and to evaluate their association with age, gender, ethnicity, and other demographic factors.MethodsIn 2012, a multi‐center cross‐sectional study was conducted in four University Hospitals in Germany. Stored frozen serum specimens obtained from vertically HIV‐infected children and adolescents were tested for antibodies against lytic and latent HHV‐8 antigens. Data on patients' demographic characteristics and medical history were recorded.ResultsA total of 214 HIV‐infected children and adolescents (105 males, 109 females) were included. The median age was 10.2 years (range 1 months–22.6 years). A high proportion of these children (62%) was born in Western Europe, whereas 65% (139/214) of their mothers were born in countries outside Western Europe. The majoritiy (91%) of the children had been treated with highly active antiretroviral therapy and 55.2% (116/210) had a HIV‐viral load<50 copies/mL. The median CD4 cell count was 1000/L (range 3–4400). The overall seroprevalence of HHV‐8 antibodies was 23.8% (51/214). Seroprevalence rates did not show significant differences between age or gender. In the group of young children aged 1 month to 35 months, 19.4% (46/31) had HHV‐8 antibodies, compared to 25% (25/100) in children aged 36 months to 11 years, and 24.1% (20/83) children 12 years and older. In the study group, seroprevalence rates were significantly lower in children who were born in Western Europe (p <0.01) compared to those born in Africa, Asia, or Eastern Europe. Clinical symptoms of HHV‐8 infection were reported to be uncommon; only one child had a history of KS at 2 years of age.ConclusionsVertically HIV‐infected pediatric patients living in Germany showed a high HHV‐8 seroprevalence of 23.8%. These rates were higher as expected in the normal pediatric population. The findings suggest that HHV‐8 infection occurred already in the first years of life.

Background Our outdoor patient clinic in Salzburg (Austria) comprises 170 HIV-infected patients. Since 2004 our laboratory has been using Cobas TaqMan 48 real time PCR analyser (Roche) for detection and quantification of HIV1 RNA. To date we could identify four patients who remained undetectable (3/4) or who presented with very low viremia (<200 copies per ml) while receiving no antiretroviral therapy at all.

Fragestellung: HIV-infizierter Schwangere erhalten inzwischen routinemässig antiretrovirale Medikamente (ART) zur vertikalen Transmissionsprophylaxe von HIV, auch wenn sie selbst keine Therapieindikation haben. Führt die Gabe dieser ART in der Schwangerschaft im Rahmen der maternofetalen Transmission zu einer Resistenzentwicklung, wodurch zukünftige Therapieoptionen HIV-infizierter Frauen reduziert werden könnten?

Fragestellung: Kommt es durch die antiretrovirale Transmissionsprophylaxe HIV-infizierter Schwangerer ohne eigene Therapieindikation zu einer Resistenzentwicklung, die deren zukünftige eigene Therapieoptionen einschränkt?

Introduction: Hypogonadism is considered to be one of the major risk factors for osteoporosis in men. Here, we sequentially studied the effects of androgen deficiency on cortical bone in aged orchiectomy (ORX) rats.Materials and Methods: One hundred seventy 13-mo-old male Fischer-344 rats were either ORX or shamoperated. After in vivo fluorochrome labeling, groups of 8-15 SHAM and ORX rats each were killed at 2 wk and 1, 2, 3, 4 6, and 9 mo after surgery. To examine the effects of testosterone replacement therapy, 9-mo-old ORX rats were supplemented with testosterone undecanoate at a weekly dose of 6 mg/kg for 4 mo. Cortical bone changes in the tibial shaft were monitored by pQCT analysis and by bone histomorphometry.Results: SHAM rats did not show age-related bone loss at the tibial diaphysis. pQCT analysis and bone histomorphometry showed cortical bone osteopenia in ORX rats, beginning from 2 mo after surgery until the end of the study. Androgen deficiency induced a sustained decrease in periosteal bone formation during the first 4 mo after ORX. However, although periosteal expansion of the tibial shaft tended to be slower in ORX rats compared with SHAM controls, the reduction in total cross-sectional area in ORX animals reached statistical significance only at 4 mo after surgery. The major mechanism for cortical bone loss in aged ORX rats was a progressive expansion of the marrow cavity, which was associated with an initial increase in endocortical eroded perimeter at 1 and 2 mo after surgery, followed by a sustained increase in endocortical bone formation until the end of the study. All these changes were prevented in aged ORX rats receiving testosterone supplementation in an insulin-like growth factor system-independent fashion.Conclusions: We conclude that androgen deficiency-induced cortical bone loss in aged, nongrowing rats is mainly caused by augmented endocortical bone remodeling.

Hintergrund: Antiretrovirale Medikamente werden bei HIV-positiven Schwangeren zur vertikalen Transmissionsprophylaxe eingesetzt. Dadurch können Resistenzen entstehen und zukünftige Therapieoptionen für die Mütter reduziert werden.

BACKGROUND:In this retrospective study the effect of antiretroviral combination regimens including the non nucleoside reverse transcriptase inhibitor (NNRTI) efavirenz (EFV) on viral load (VL) and CD4-cell count in heavily pretreated HIV-infected children was investigated.DESIGN:The data of 15 children (< 16 years) were evaluated during a treatment period of at least 52 weeks. Patients received a median of 4 prior antiretroviral regimens and were changed to combinations with EFV because of renewed increasing VL.METHODS:Viral load (Amplicor, detection limit 50 copies/ml) and CD4-cells were measured every 4-8 weeks.RESULTS:The median reduction of the viral load for the total study population was 1.9 log10 (0.8 - 4.7), 2.3 log(10) (0.5 - 4.7) and 2.6 log(10) (0-4.7) after 12, 24 and 52 weeks, respectively. After 24 weeks 7/15 children and after 52 weeks 9/15 patients had reductions of the VL below the detection limit. The median increase of CD4-cells in the study population during the treatment period were 104 cells/microl (189-969), 220 cells/microl (170-831) and 321 cells/ml (162-574) after 12, 24 and 52 weeks, respectively.CONCLUSIONS:In children with intensive prior antiretroviral therapy and multiple therapeutic failures with PI-containing regimens, combination therapy including EFV resulted in an excellent antiretroviral efficacy. After 52 weeks 9/15 patients had persistent reductions of the VL below 50 copies/ml, although none of these children reached this level of viral suppression during their multiple prior protease inhibitor-containing regimens.

The effect of cAMP on the transcriptional activity of the HIV‐1 long terminal repeat/enhancer was investigated and compared to the effect of cAMP on virus replication. In culture cAMP repressed virus replication in vivo using different cell types. Transient transfection studies with HIV‐1 enhancer‐derived luciferase reporter gene constructs identified the minimal DNA sequence mediating the negative regulatory effect of cAMP on HIV‐1 transcription. A single nuclear factor κB element from the HIV‐1 enhancer mediates the repressive effect on transcription. AP‐2 is not involved in cAMP repression. Stable transfection of Jurkat T cells with the co‐activators CREB binding protein (CBP) and p300 completely abolished the cAMP repressive effect, supporting the hypothesis that elevation of intracellular cAMP increases phosphorylation of CREB, which then competes with phosphorylated p65 and Ets‐1 for limiting amounts of CBP/p300 thereby mediating the observed repressive effect on transcription. These findings suggest an important role of cAMP on HIV‐1 transcription.

HIV is transmitted mainly by blood and sexual contact. Nosocomial infections occur most frequently by needle sticks or by contamination of eczematous skin lesions. The conjunctiva of the eye had been suggested to be a potential site of entry of HIV. This report presents evidence on the method of HIV transmission through the spillage of small amounts of contaminated serum into one eye. This mode of HIV transmission can be easily prevented by protecting the eyes with glasses and goggles. A blood sample from a 26-year-old male laboratory technician, when tested for antibodies to HIV, showed a moderately high extinction in the screening enzyme-linked immunosorbent assay (ELISA; Abbott Laboratories, North Chicago, IL, USA; sample/cut-off ratio 2.8) and p24 and gp160 bands in the HIV-1 immunoblot, a pattern suggesting early seroconversion. A sample taken 1 week later showed high reactivity in two ELISA (Abbott and Dade–Behring, Marburg, Germany; sample/cut-off ratio > 8) and p24, p55 and gp160 immunoblot bands. A follow-up sample taken 1 month after the first bleed showed the same high ELISA reactivity, but all bands in the immunoblot, indicating unambiguous seroconversion. The technician reported an accident approximately 5 weeks before the first specimen was drawn, when after centrifugation he was opening the stopper of a vacutainer tube by hand, and not using the tool supplied for that purpose. He was wearing gloves during this manipulation. He felt a droplet spill into his left eye, to which he reacted by blinking rapidly and not by washing the eye. At the time of the accident he was wearing contact glass lenses, which had induced a moderate conjunctivitis. He did not report the accident, nor was the number of the specimen identified. It was possible to trace the source of the infection in the small hospital where he was working because at that time only one HIV-infected patient was present. This was a Thai women, who had been hospitalized after a severe traffic accident and had been treated in the intensive care unit for more than 1 month. At the time of hospitalization the CD4 cell count of the patient was 318/μl and viral load was 40 copies/ml, determined using a test that was probably not suitable to quantitate the HIV-1E virus accurately [1]. She was not receiving antiretroviral therapy because her HIV infection was not known before the traffic accident. The technician had no access to the intensive care unit and denied having had any sexual contact with the patient. Nucleic acid sequence analysis of the C2V3 region of the HIV env gene from the patient and the technician by nested polymerase chain reaction (PCR), using the primers 5tat14 and 3env 155 in the first round and 5env72 and 3ed33 in the second round, revealed a sequence that clustered in the HIV-1E subtypes from Thailand when performed by computer analysis with the Treecon program as shown in Fig. 1[2]. Within the 350 nucleotides of the sequences of the patient (mvp7049–98) and technician two base pairs were different, leading to two amino acid mutations: glycine (GGT) to serine (AGT) and histidine (CAT) to proline (CCT) in the V3 loop of the technician's HIV (mvp7050–98). Re-analysis of the HIV nucleic acid sequence of the technician in a blood sample drawn after 3 weeks gave an identical sequence (mvp7581–98). Molecular analysis thus showed that both viruses were closely related, indicating that the technician harboured the HIV-1E of the Thai patient.Fig. 1.: Evolutionary tree of randomly selected HIV-1E viruses including the sequences of the index patient and the technician. It can easily be seen that the HIV of the Thai women (mvp7049–98) and the HIV of the technician (mvp7050 and 7581–98) cluster closely together.The amino acid sequence of the V3 loop was as follows: mvp7049–98 CTRPSNNTRTGIHMGPGQVFYRTGEIIGDIRKAYC emvp7050–98 CTRPSNNTRTSIPMGPGQVFYRTGEIIGDIRKAYC When the technician was interviewed in order to exclude other routes of HIV infection, he reported only one homosexual contact one year before the accident. From data available to us, HIV-1E is not present in the homosexual community in Munich, and acquisition of this HIV-1E virus by the technician through sexual contact is highly improbable. The C2V3 region sequence analysis and position in the evolutionary tree of HIV-1E documents the similarity between the two viruses, and the time course of the technician's infection indicates that the HIV-1E of the Thai woman was the source of his infection. Because no needle stick or other laboratory accident was reported by the technician, the only plausible manner of HIV transmission was via the reported serum droplet into his eye. During the past 10 years our laboratory has investigated seven spillages of blood from AIDS patients into the eye, mainly connected with venepuncture. All healthcare workers involved immediately rinsed the eye with tap water and one started antiretroviral therapy; none acquired an HIV infection. Why the technician did not rinse the eye after the accident is unclear, because he had been instructed twice within the previous 6 months about immediate action after eye contamination. The possible transmission of HIV by contamination of the conjunctiva has been suspected previously as the mode of infection of a nurse in Italy [3]. Reports have also indicated that HIV can be cultured from cornea cells [4] and isolated from tears [4,5]. Our report indicates that after prolonged exposure with a sufficient quantity of virus, the mucous membrane of the eye seems to be susceptible to HIV entry. In this case viral entry might have been facilitated by the inflamed conjunctival cells. Protection of the eye can easily stop this rare mode of HIV transmission. Josef Eberlea Jürgen Habermannb Lutz G. Gürtlerc

Reactivation of Chronic Hepatitis C Virus Infection by Immunoadsorption in Factor VIII Inhibitor Haemophilia -

HIV-1 group O has its epicenter in Cameroon and neighboring countries and is responsible for 3 to 5% of all HIV infections in this region. It is believed that HIV-1 group O was introduced into the human population by a separate cross-species transmission, occurring independently of the HIV-1 (group M and group N) and HIV-2 transmissions. We have studied the coreceptor requirements of 12 primary HIV-1 O-type isolates from individuals with different clinical symptoms. Only 2 of these 12 viruses showed a syncytium-inducing phenotype after infection of primary peripheral blood mononuclear cells (PBMCs) and were infectious for the T cell line C8166. These isolates used CXCR4 as a coreceptor for entry, whereas the remaining isolates used only CCR5 efficiently. One isolate was able to use BOB and CCR8 as coreceptors in addition to CXCR4. All group O isolates tested were efficiently inhibited by SDF-1 or RANTES, the natural ligands of CXCR4 and CCR5, respectively. These results indicate that CXCR4 and CCR5 are the principal coreceptors for HIV1 O-type viruses. Most of the HIV-1 group O isolates studied were derived from patients at later stages of the disease. Although HIV-1 group O and group M infections do not differ in their pathogenesis, the studied isolates did not evolve to use a broad range of coreceptors as described for HIV-1 group M and HIV-2.

A 35-year-old female health worker with some occupational risk for acquisition of HIV-1 infection had an acute infectious mononucleosis-like syndrome. She was in hospital for several days. 3 weeks later, she was positive for HIV-1 antibodies (Abbott HIV-1/HIV-2 3rd generation plus EIA). In an HIV-1 immunoblot (BioRad NovaPath TM HIV-1 Immunoblot) there was reactivity against viral proteins p17, p24, p55, p120, and p160. Quantitative analysis of viral RNA load (Organon Teknika, NASBA HIV-1 RNA QT) yielded 45 000 copies per mL. Treatment was started with zidovudine and lamivudine. Viral load rapidly declined and monitoring showed values around 160 copies per mL. Humoral immune response against HIV-1 antigens meanwhile proceeded, she has now been free from symptoms for 18 months. She initially denied any risk factors in her private life, but later disclosed that 3 weeks before her illness she had unsuccessfully undergone artifical insemination with fresh sperm. Upon notification of the patient’s illness, the gynaecologist concerned retested the donor, who had meanwhile seroconverted. Viral RNA from the recipient’s serum (collected 12 weeks after the date of insemination) was reverse transcribed and the V3 region was amplified by nested PCR. Nucleotide sequence determination of the amplification product revealed 100% identity with viral sequences from the donor on the nucleotide level which was amplified from the DNA of peripheral blood mononuclear cells. Physicians must be discouraged from using fresh sperm. The early—highly viraemic, seronegative—stage of HIV-1 infection may carry a high risk of infection, especially since seminal viral load parallels viraemia. As long as quarantine storage of anonymous sperm donation is not mandatory in all countries (as it is not in Germany), artificial insemination still has to be taken into consideration as a source of HIV-1 infection.

We investigated HIV-1 diversity by means of heteroduplex mobility assay (HMA) genotyping. We studied 199 samples from patients originating from 26 countries and living in France. The HMA successfully genotyped 182 (91%) of these samples, as follows: 77 (42%) subtype A, 57 (31%) subtype B, 5 (3%) subtype C, 5 (3%) subtype D, 8 (4%) subtype E, 22 (12%) subtype F, 5 (3%) subtype G, and 3 (2%) subtype H. We were not able to genotype 12 samples by means of the HMA. These latter strains were sequenced, and phylogenetic analyses revealed that they were highly divergent subtype A-, D-, or G-related strains. Eight (of 12) subtype D strains were indeterminate by HMA, owing to the broad intrasubtype diversity, suggesting that new reference subtype D plasmids are required, as previously proposed. Thirty-seven strains belonging to the different subtypes were sequenced, and the results showed perfect concordance with the HMA results. Interlaboratory quality controls confirmed the reliability of the HMA for HIV-1 subtyping, despite the extensive viral variability. However, plasmid selection must be continuously revised to cover viral diversification.