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We describe 10 patients with severe coronavirus disease 2019 (COVID-19) who received tocilizumab and dexamethasone. We correlated isolation duration with cycle thresholds (Ct) values of nucleic acid amplification tests, clinical state and viral cultures. Isolation duration exceeded 21 days for 7 patients due to positive viral cultures or Ct values

The objective of this study was to validate the use of spring water gargle (SWG) as an alternative to oral and nasopharyngeal swab (ONPS) for SARS‐CoV‐2 detection with a laboratory‐developed test. Healthcare workers and adults from the general population, presenting to one of two COVID‐19 screening clinics in Montréal and Québec City, were prospectively recruited to provide a gargle sample in addition to the standard ONPS. The paired specimens were analyzed using thermal lysis followed by a laboratory‐developed nucleic acid amplification test (LD‐NAAT) to detect SARS‐CoV‐2, and comparative performance analysis was performed. An individual was considered infected if a positive result was obtained on either sample. A total of 1297 adult participants were recruited. Invalid results (n = 18) were excluded from the analysis. SARS‐CoV‐2 was detected in 144/1279 (11.3%) participants: 126 from both samples, 15 only from ONPS, and 3 only from SWG. Overall, the sensitivity was 97.9% (95% CI: 93.7–99.3) for ONPS and 89.6% (95% CI: 83.4–93.6; p = 0.005) for SWG. The mean ONPS cycle threshold (Ct) value was significantly lower for the concordant paired samples as compared to discordant ones (22.9 vs. 32.1; p, Highlights Using a laboratory‐developed NAAT preceded by thermal lysis, the overall percent agreement between spring water gargle (SWG) and oral combined with nasopharyngeal swab (ONPS), sampled at the same time among 1297 participants, is excellent (98.6%).Although the SARS‐CoV‐2 NAAT from SWG is globally less sensitive than from ONPS (89.6% vs. 97.9%), the difference is markedly less in individuals symptomatic for

Aerococcus urinae is a urinary pathogen with well-described resistance to fluoroquinolones. This study aimed to validate the gradient diffusion (GD) method (Etest) on cation-adjusted Mueller-Hinton agar with 5% sheep blood for testing the susceptibilities of Aerococcus urinae to the antimicrobial agents ciprofloxacin and levofloxacin and to compare the Etest to the broth microdilution (BMD) method from CLSI document M45-A3. Agar dilution (AD), as recommended by EUCAST, was used as an alternative reference method to arbitrate discrepancies or address technical issues. Aerococcus urinae isolates from urinary specimens were prospectively collected between June 2016 and December 2017 from six hospitals in Quebec, Canada, and identifications were confirmed using Vitek MS with the IVD 3.0 database. Of the 207 isolates tested using BMD, 37 (17.9%) showed trailing and 19 (9.2%) showed insufficient growth; these were tested using AD. Also, 38 isolates (18.4%) for ciprofloxacin and 13 isolates (6.3%) for levofloxacin showed a lack of essential or categorical agreement between the Etest and BMD and were also tested by AD. By use of a combined reference method (BMD or AD), the susceptibility rates of Aerococcus urinae were 82.6% and 81.6% for ciprofloxacin and levofloxacin, respectively. Categorical agreement between GD and the combined reference methods was 95.2% for ciprofloxacin and 97.1% for levofloxacin, with no very major error identified. Major and minor error rates were 0.6% and 4.3% for ciprofloxacin and 1.2% and 1.9% for levofloxacin. Overall, antimicrobial susceptibility testing (AST) using the Etest on sheep blood agar showed good agreement with the reference methods and can be considered by clinical laboratories wishing to perform AST on Aerococcus urinae isolates.

Background Nasopharyngeal swab has long been considered the specimen of choice for the diagnosis of respiratory viral infections, including SARS-CoV-2 infection, but it suffers from several drawbacks: its discomfort limits screening acceptability, and it is vulnerable to shortages in both specialized materials and trained healthcare workers in the context of a pandemic. Methods We prospectively compared natural spring water gargle to combined oro-nasopharyngeal swab (ONPS) for the diagnosis of coronavirus disease 2019 (COVID-19) in paired clinical specimens (1005 ONPS and 1005 gargles) collected from 987 unique early symptomatic as well as asymptomatic individuals from the community. Results Using a direct RT-PCR method with the Allplex™ 2019-nCoV Assay (Seegene), the clinical sensitivity of the gargle was 95.3% (95% confidence interval [CI], 90.2 – 98.3%), similar to the sensitivity of the ONPS (93.8%; 95% CI, 88.2 – 97.3%), despite significantly lower viral RNA concentration in gargles, as reflected by higher cycle threshold values. No single specimen type detected all COVID-19 cases. SARS-CoV-2 RNA was stable in gargles at room temperature for at least 7 days. Conclusion The simplicity of this sampling method coupled with the accessibility of spring water are clear advantages in a pandemic situation where testing frequency, turnaround time and shortage of consumables and trained staff are critical elements.

We have developed a new method for the identification of bacterial species causing Urinary Tract Infections. The first training step used DIA analysis on multiple replicates of bacterial inoculates to define a peptide signature by machine learning classifiers. In a second identification step, the signature is monitored by targeted proteomics on unknown samples. This fast, culture-free and accurate method paves the way of the development of new diagnostic approaches limiting the emergence of antimicrobial resistances., Graphical Abstract Highlights Fast and culture-free method for the identification of the 15 bacterial species causing UTIs. Combination of DIA analysis and machine learning algorithms to define a peptide signature. High accuracy, good linearity and reproducibility, sensitivity below standard threshold. Transferability to other laboratories and other mass spectrometers., Fast identification of microbial species in clinical samples is essential to provide an appropriate antibiotherapy to the patient and reduce the prescription of broad-spectrum antimicrobials leading to antibioresistances. MALDI-TOF-MS technology has become a tool of choice for microbial identification but has several drawbacks: it requires a long step of bacterial culture before analysis (≥24 h), has a low specificity and is not quantitative. We developed a new strategy for identifying bacterial species in urine using specific LC-MS/MS peptidic signatures. In the first training step, libraries of peptides are obtained on pure bacterial colonies in DDA mode, their detection in urine is then verified in DIA mode, followed by the use of machine learning classifiers (NaiveBayes, BayesNet and Hoeffding tree) to define a peptidic signature to distinguish each bacterial species from the others. Then, in the second step, this signature is monitored in unknown urine samples using targeted proteomics. This method, allowing bacterial identification in less than 4 h, has been applied to fifteen species representing 84% of all Urinary Tract Infections. More than 31,000 peptides in 190 samples were quantified by DIA and classified by machine learning to determine an 82 peptides signature and build a prediction model. This signature was validated for its use in routine using Parallel Reaction Monitoring on two different instruments. Linearity and reproducibility of the method were demonstrated as well as its accuracy on donor specimens. Within 4h and without bacterial culture, our method was able to predict the predominant bacteria infecting a sample in 97% of cases and 100% above the standard threshold. This work demonstrates the efficiency of our method for the rapid and specific identification of the bacterial species causing UTI and could be extended in the future to other biological specimens and to bacteria having specific virulence or resistance factors.

Herpes simplex virus (HSV) DNA polymerase (Pol) mutations can confer resistance to all currently available antiherpetic drugs. However, discrimination between mutations responsible for drug resistance and those that are part of viral polymorphism can be difficult with current methodologies. A new system is reported for rapid generation of recombinant HSV type 1 (HSV-1) DNA Pol mutants based on transfection of a set of overlapping viral cosmids and plasmids. With this approach, twenty HSV-1 recombinants with single or dual mutations within the DNA pol gene were successfully generated and subsequently evaluated for their susceptibilities to acyclovir (ACV), foscarnet (FOS), cidofovir (CDV), and adefovir (ADV). Mutations within DNA Pol conserved regions II (A719T and S724N), VI (L778M, D780N, and L782I), and I (F891C) were shown to induce cross-resistance to ACV, FOS, and ADV, with two of these mutations (S724N and L778M) also conferring significant reduction in CDV susceptibility. Mutant F891C was associated with the highest levels of resistance towards ACV and FOS and was strongly impaired in its replication capacity. One mutation (D907V) lying outside of the conserved regions was also associated with this ACV-, FOS-, and ADV-resistant phenotype. Some mutations (K522E and Y577H) within the δ-region C were lethal, whereas others (P561S and V573M) induced no resistance to any of the drugs tested. Recombinants harboring mutations within conserved regions V (N961K) and VII (Y941H) were resistant to ACV but susceptible to FOS. Finally, mutations within conserved region III were associated with various susceptibility profiles. This new system allows a rapid and accurate evaluation of the functional role of various DNA Pol mutations, which should translate into improved management of drug-resistant HSV infections.

Nucleoside analogues such as acyclovir and ganciclovir have been the mainstay of therapy for alphaherpesviruses (herpes simplex virus (HSV) and varicella-zoster virus (VZV)) and cytomegalovirus (CMV) infections, respectively. Drug-resistant herpesviruses are found relatively frequently in the clinic, almost exclusively among severely immunocompromised patients receiving prolonged antiviral therapy. For instance, close to 10% of patients with AIDS receiving intravenous ganciclovir for 3 months excrete a drug-resistant CMV isolate in their blood or urine and this percentage increases with cumulative drug exposure. Many studies have reported that at least some of the drug-resistant herpesviruses retain their pathogenicity and can be associated with progressive or relapsing disease. Viral mutations conferring resistance to nucleoside analogues have been found in either the drug activating/phosphorylating genes (HSV or VZV thymidine kinase, CMV UL97 kinase) and/or in conserved regions of the viral DNA polymerase. Currently available second line agents for the treatment of herpesvirus infections--the pyrophosphate analogue foscarnet and the acyclic nucleoside phosphonate derivative cidofovir--also inhibit the viral DNA polymerase but are not dependent on prior viral-specific activation. Hence, viral DNA polymerase mutations may lead to a variety of drug resistance patterns which are not totally predictable at the moment due to insufficient information on specific drug binding sites on the polymerase. Although some CMV and HSV DNA polymerase mutants have been found to replicate less efficiently in cell cultures, further research is needed to correlate viral fitness and clinical outcome.

The microbicidal activity of sodium lauryl sulfate (SLS) against human immunodeficiency virus type 1 (HIV-1) was studied in cultured cells. Pretreatment of HIV-1NL4-3with SLS decreased, in a concentration-dependent manner, its infectivity when using 1G5 as target cells. In the absence of a viral pretreatment period or when 1G5 cells were pretreated with SLS, the surfactant-induced inactivation of viral infectivity was less pronounced, especially at concentrations between 375 and 550 μM. SLS had no effect on HIV-1 when the virus was adsorbed to 1G5 cells by a 2-h incubation period. SLS almost completely inhibited the fusion process by decreasing the attachment of HIV-1 to target cells. SLS also inhibited the infectivity of HIV-1-based luciferase reporter viruses pseudotyped with the amphotropic murine leukemia virus envelope (which enters cells in a CD4-, CCR5-, and CXCR4-independent manner), indicating that SLS may inactivate other envelope viruses. In contrast, no effect was seen with vesicular stomatitis virus envelope glycoprotein G (which enters cells through receptor-mediated endocytosis) pretreated with up to 700 μM SLS. SLS also decreased, in a dose-dependent manner, the HIV-1-dependent syncytium formation between 1G5 and J1.1 cells after a 24-h incubation. The reduction of luciferase activity was more pronounced when J1.1 cells (which express HIV-1 proteins on their surface) were pretreated with SLS rather than 1G5 cells. Taken together, our results suggest that SLS could represent a candidate of choice for use in vaginal microbicides to prevent the sexual transmission of HIV and possibly other pathogens causing sexually transmitted diseases.

The ability of liposomes bearing anti-HLA-DR Fab′ fragments at the end termini of polyethyleneglycol chains (sterically stabilized immunoliposomes) to target HLA-DR expressing cells and increase the accumulation of liposomes into lymphoid organs has been evaluated and compared to that of conventional liposomes, sterically stabilized liposomes and conventional immunoliposomes after a single subcutaneous injection to mice. The accumulation of sterically stabilized liposomes in lymph nodes was higher than that of conventional liposomes. Sterically stabilized immunoliposomes accumulated much better than conventional immunoliposomes in all tissues indicating that the presence of PEG has an important effect on the uptake of immunoliposomes by the lymphatic system. Fluorescence microscopy studies showed that sterically stabilized liposomes are mainly localized in macrophage-rich areas such as the subcapsular region of lymph nodes and in the red pulp and marginal zone of the spleen. In contrast, sterically stabilized immunoliposomes mostly accumulated in the cortex in which follicles are located and in the white pulp of the spleen. As the human HLA-DR determinant of the major histocompatibility complex class II is expressed on activated CD4+ T lymphocytes and antigen presenting cells such as monocyte/macrophages and dendritic cells, known as the cellular reservoirs of HIV-1, liposomes bearing anti-HLA-DR antibodies constitute an attractive approach to concentrate drugs in HIV-1 reservoirs and improve their therapeutic effect.

The ability of liposomes bearing anti-HLA-DR Fab′ fragments to target cells expressing the human HLA-DR determinant of the major histocompatibility complex class II (MHC-II) has been evaluated and compared to that of conventional liposomes. Anti-HLA-DR immunoliposomes did not bind to HLA-DR-negative cells. In contrast, a high level of binding was observed following incubation of immunoliposomes with cells bearing important levels of human HLA-DR. The accumulation of conventional and murine anti-HLA-DR immunoliposomes in different tissues has been investigated following a single subcutaneous injection given in the upper back of C3H mice. Anti-HLA-DR immunoliposomes resulted in a much better accumulation in the cervical and brachial lymph nodes when compared to conventional liposomes. The accumulation in the liver was similar for both liposomal preparations, whereas an approximately twofold decrease in accumulation was observed for immunoliposomes in the spleen. Given that HLA-DR surface marker is expressed on monocyte/macrophages and activated CD4+ T lymphocytes, the primary cellular reservoirs of the human immunodeficiency virus (HIV), the use of liposomes bearing surface-attached anti-HLA-DR could constitute a convenient strategy to more efficiently treat this debilitating retroviral disease. Moreover, the reported incorporation of high amounts of host-encoded HLA-DR proteins by HIV particles renders the use of liposomes bearing anti-HLA-DR antibodies even more attractive.

Sequential herpes simplex virus (HSV) isolates from AIDS patients receiving foscarnet therapy were evaluated for susceptibility to adefovir. Foscarnet-resistant isolates with DNA polymerase mutations in regions II, VI, and between I and VII were also associated with an important decrease in susceptibility to adefovir (mean IC50 increase: 4.6-fold compared to pre-foscarnet or wild-type isolates) suggesting that adefovir-resistant HSV could be selected in vivo by foscarnet therapy. J. Med. Virol. 67:88–91, 2002. © 2002 Wiley-Liss, Inc.

Objective To evaluate the ability of liposomes bearing anti-HLA-DR Fab' fragments (immunoliposomes) and containing amphotericin B (AmB) to target and neutralize cell-free HIV-1 particles and virally-infected cells. Methods The effect of AmB on the attachment and fusion of HIV-1(NL4-3) to Jurkat E6.1 cells has been evaluated using a p24 enzymatic assay. The ability of AmB to inhibit HIV-1-based luciferase reporter viruses pseudotyped with HXB2, AML-V and VSV-G envelopes has been evaluated in Jurkat E6.1 cells. The efficacy of free and immunoliposomal AmB to inhibit cell-free HIV, that have incorporated or not HLA-DR molecules, has been evaluated in HLA-DR/negative (NEG) 1G5 T cells and HLA-DR/positive (POS) Mono Mac 1 cells. Results AmB inhibited HIV infectivity independently of the nature of viral envelope proteins. Pretreatment of HIV with AmB had no major effect on viral attachment and fusion process to Jurkat E6.1 cells. Immunoliposomal AmB (0.5 microg/ml) led to a 77% inhibition of replication of HLA-DR/POS HIV-1 with no cell toxicity, whereas free AmB had no significant antiviral activity at this concentration. A complete inhibition of viral replication was observed following incubation of viruses with immunoliposomal AmB (2.5 microg/ml). Anti-HLA-DR immunoliposomes containing AmB had no effect on the infectivity of HLA-DR/NEG HIV-1 particles in HLA-DR/NEG T lymphoid cells but completely inhibited replication of viruses in an HLA-DR/POS monocytic cell line. Conclusion The incorporation of neutralizing agents in anti-HLA-DR immunoliposomes could represent a novel therapeutic strategy to specifically target cell-free HIV particles and virally-infected cells to treat HIV infection more efficiently.

The efficacy of sodium lauryl sulfate (SLS), a sulfated anionic chaotropic surfactant, and dextran sulfate (DS), a polysulfated carbohydrate, against herpes simplex virus (HSV) and human immunodeficiency virus (HIV) infections was evaluated in cultured cells and in different murine models of HSV infection. Results showed that both SLS and DS were potent inhibitors of the infectivities of various HSV-1 and HSV-2 strains. Pretreatment of HIV-1 (strain NL4-3) with SLS also reduced its infectivity to 1G5 cells. DS prevented the binding of HSV to cell surface receptors and therefore its entry into cells. Pretreatment of HSV-1 (strain F) with 50 μM SLS resulted in a complete loss of virus infectivity to Vero cells. However, viruses were able to enter into cells and to produce in the nuclei capsid shells devoid of a DNA core. The amount of the glycoprotein D gene produced in these cells remained unchanged compared to controls, suggesting that SLS could interfere with the maturation of the virus. At a higher SLS concentration (100 μM), HSV was highly damaged by SLS pretreatment and only a few viral particles could enter into cells to produce abnormal capsids. Although DS was a more potent inhibitor of HSV infectivity in vitro, it was unable to provide any protection in murine models of HSV infection. However, SLS conferred a complete protection of animals infected cutaneously with pretreated viruses. In addition, skin pretreatment of mice with a polymer formulation containing SLS completely prevented the development of cutaneous lesions. More interestingly, intravaginal pretreatment of mice with SLS in a buffered solution also completely protected against lethal HSV-2 infection. Taken together, our results suggest that SLS could thus represent a candidate of choice as a microbicide to prevent the sexual transmission of HIV, HSV, and possibly other pathogens that cause sexually transmitted diseases.