bioMérieux.

SummaryThe viral regulatory gene, nef, is unique to the human immunodeficiency viruses (HIV) and their related primate lentiviruses. Expression of the nefgene has been shown to be essential to the maintenance of high levels of virus replication and the development of pathogenesis in the animal model of simian immunodeficiency virus (SIV) infection. In contrast to this in vivo model, the use of standard T cell culture systems to study neffunction in vitro has produced a spectrum of contradictory results, and has failed to demonstrate a significant positive influence of nefon viral life cycle. We have developed a cell model to study regulation of HIV-1 replication that we believe reflects more accurately virus-cell interactions as they occur in vivo. Our experimental system used acute virus infection of purified, quiescent CD4 lymphocytes and subsequent induction of viral replication through T cell activation. With this cell model, NL4-3 virus clones with open and mutated nef reading frames were compared for replication competence. The clones with nefmutations showed reproducible and significant reductions in both rates of growth and maximal titers achieved. The degree of reduced replication was dependent on initial virus inoculum and the timing of T cell activation. The influence of nef was highly significant for induction of virus replication from a latent state within resting CD4 cells. Its effect was less apparent for virus infection of fully proliferating CD4 cells. This study demonstrates that nefconfers a positive growth advantage to HIV-1 that becomes readily discernable in the primary cell setting of virus induction through T cell activation. The experimental cell model, which we describe here, provides not only a means to study neffunction in vitro, but also provides important clues to the function of nef in HIV infection in vivo.

Objectives mRNA COVID-19 vaccines have shown high effectiveness in the prevention of symptomatic COVID-19, hospitalization, severe disease, and death. Nevertheless, a minority of vaccinated individuals might get infected and suffer significant morbidity. Characteristics of vaccine breakthrough infections have not been studied. We sought to portray the population of Israeli patients, who were hospitalized with COVID-19 despite full vaccination. Methods A retrospective multicenter cohort study of 17 hospitals included Pfizer/BioNTech's BNT162b2 fully-vaccinated patients who developed COVID-19 more than 7 days after the second vaccine dose and required hospitalization. The risk for poor outcome, defined as a composite of mechanical ventilation or death, was assessed. Results 152 patients were included, accounting for half of hospitalized fully-vaccinated patients in Israel. Poor outcome was noted in 38 patients and mortality rate reached 22% (34/152). Notable, the cohort was characterized by a high rate of comorbidities predisposing to severe COVID-19, including hypertension (108, 71%), diabetes (73, 48%), CHF (41, 27%), chronic kidney and lung diseases (37, 24% each), dementia (29, 19%), and cancer (36, 24%), and only 6 (%) had no comorbidities. Sixty (40%) of the patients were immunocompromised. Higher SARS-CoV-2 viral-load was associated with a significant risk for poor outcome. Risk also appeared higher in patients receiving anti-CD20 treatment and in patients with low titers of anti-spike IgG, but these differences did not reach statistical significance. Conclusions We found that severe COVID-19 infection, associated with a high mortality rate, might develop in a minority of fully-vaccinated individuals with multiple comorbidities. Our patients had a higher rate of comorbidities and immunosuppression compared to previously reported non-vaccinated hospitalized COVID-19 patients. Further characterization of this vulnerable population may help to develop guidance to augment their protection, either by continued social-distancing, or by additional active or passive vaccinations.

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The replication competence of human immunodeficiency virus type 1 genomes containing mutations in the nefopen reading frame was evaluated in continuous cell lines. Mutants that contained a deletion in the nefopen reading frame, premature termination codons, or missense mutations in the N-terminal myristoylation signal were constructed. The replication of these mutants was tested in three ways. First, plasmid genomes were used to transfect T-lymphoblastoid cells. Second, low-passage posttransfection supernatants were used to infect cells with a relatively low virus input. Third, high-titer virus stocks were used to infect cells with a relatively high virus input. These experiments demonstrated a 100to 10,000-fold decrement in p24 production by the nef mutants compared with that by the wild-type virus. The greatest difference was obtained after infection with the lowest virus input. The myristoylation signal was critical for this positive elfect of nef. To investigate the mechanism of the positive influence of nef, nef-positive and nef-minus viruses were compared during a single cycle of replication. These single-cycle experiments were initiated both by infection with high-titer virus stocks and by transfection with viral DNA. Single-cycle infection yielded a threeto fivefold decrement in p24 production by nef-minus virus. Single-cycle transfection yielded equal amounts of p24 production. These results implied that nef does not affect replication after the provirus is established. In support of these results, viral production from cells chronically infected with nef-positive or nef-minus viruses was similar over time. To determine whether the effect of nef was due to infectivity, end point titrations of nef-positive and nef-minus viruses were performed. nef-positive virus had a greater infectivity per picogram of HIV p24 antigen than nef-minus virus. These data indicated that the positive influence of nef on viral growth rate is due to an infectivity advantage of virus produced with an intact nef gene.

Many practical clinical questions regarding the management of human immunodeficiency virus (HIV)-associated neurocognitive disorder (HAND) remain unanswered. We sought to identify and develop practical answers to key clinical questions in HAND management. Sixty-six specialists from 30 countries provided input into the program, which was overseen by a steering committee. Fourteen questions were rated as being of greatest clinical importance. Answers were drafted by an expert group based on a comprehensive literature review. Sixty-three experts convened to determine consensus and level of evidence for the answers. Consensus was reached on all answers. For instance, good practice suggests that all HIV patients should be screened for HAND early in disease using standardized tools. Follow-up frequency depends on whether HAND is already present or whether clinical data suggest risk for developing HAND. Worsening neurocognitive impairment may trigger consideration of antiretroviral modification when other causes have been excluded. The Mind Exchange program provides practical guidance in the diagnosis, monitoring, and treatment of HAND.

Recent data on the phenotype of nef-defective HIV-1 in vitro indicate a new function of the Nef gene product: enhancement of viral infectivity. Single-cycle replication studies have suggested that Nef enhances the efficiency of an early step during viral replication, a step that leads to the establishment of viral DNA. To test this interpretation, the accumulation of low-molecular-weight (unintegrated) viral DNA was measured in cells following exposure to wild-type and nef-defective viruses. nef-defective virus accumulated less DNA than the wild type. This difference was observed after as little as 5 hr of exposure to virus. However, the reverse transcriptase activities of wild-type and nef-defective viruses were equal when measured in cell-free assays using either exogenous or endogenous templates. In addition, the abilities of these viruses to bind and enter cells were not significantly different. Together, these data suggest that Nef optimizes postentry events that are required for efficient synthesis of viral DNA. To determine if these effects were related to the property of Nef-mediated downregulation of CD4, growth curves of these viruses were determined using cells that express a CD4 molecule unable to respond to Nef. nef-defective virus remained attenuated in these cells, indicating that Nef-mediated downregulation of CD4 is not required for Nef-mediated enhancement of viral propagation in vitro.

Candida species have emerged as frequent causes of nosocomial bloodstream infection (BSI) in association with well-defined risk factors, including prolonged hospitalization, abdominal surgery, antibiotic treatment, neutropenia and central venous catheterization (14). Candidemia is associated with high rates of attributable mortality, prolongation of hospital stay, and excessive costs (28). In recent years, there has been a shift in the distribution of Candida species causing invasive infection, with non-albicans species now surpassing Candida albicans in many institutions (14,25). Of particular concern is the rising incidence of the azolenonsusceptible species C. glabrata and the inherently fluconazoleresistant species C. krusei (11,25,27).Fluconazole is often used as empirical treatment of candidemia. However, given the correlation between the survival rate and the timely initiation of appropriate treatment for candidemia (8), accurate assessment of the risk of fluconazole-resistant Candida (FRC) BSI is of prime importance. Patients who were recently treated with an azole drug are at increased risk of infection with FRC (9) and should be treated initially with an echinocandin agent according to current guidelines (18). However, experimental and clinical data support the notion that nonantifungal antimicrobial agents also affect the risk of colonization and infection with FRC (15,17,22). Since exposure to antibacterial drugs among at-risk patients far exceeds exposure to antifungal agents, even modest effects of individual antibacterials could translate into significant overall changes in the susceptibility patterns of Candida spp. Nevertheless, the collateral effects of antibacterial drugs on Candida spp. are poorly understood. To address this question, we analyzed prospectively collected data from a nationwide study of candidemia in Israel and examined the association between exposure to antifungal and antibacterial agents and the risk of infection with FRC. MATERIALS AND METHODS Study design.We performed a prospective nationwide study of candidemia in Israel from November 2005 through June 2007. Eighteen medical centers, which together account for 75% of the hospital beds in Israel, were included. All candidemia episodes that occurred in the participating centers during the study period were eligible for inclusion in this study. Clinical data were prospectively entered into standardized data forms by on-site investigators at each of the centers. The Candida sp. clinical isolates underwent preliminary identification and susceptibility testing in each center according to local practices. Subsequently, the isolates were transferred together with the corresponding data forms to the central study site, where species identification and susceptibility testing were performed as detailed below. The data forms were collected by the study coordinator, reviewed by the principal investigator, and entered into a computerized database. The study was approved by the ethics committee of each of the participating centers.Data...