Frequent Cross-Resistance to Dapivirine in HIV-1 Subtype C-Infected Individuals after First-Line Antiretroviral Therapy Failure in South Africa.

A vaginal ring containing dapivirine (DPV) has shown moderate protective efficacy against HIV-1 acquisition, but the activity of DPV against efavirenz (EFV)- and nevirapine (NVP)-resistant viruses that could be transmitted is not well defined. We investigated DPV cross-resistance of subtype C HIV-1 from individuals on failing NVP- or EFV-containing antiretroviral therapy (ART) in South Africa. Plasma samples were obtained from individuals with >10,000 copies of HIV RNA/ml and with HIV-1 containing at least one non-nucleoside reverse transcriptase (NNRTI) mutation. Susceptibility to NVP, EFV, and DPV in TZM-bl cells was determined for recombinant HIV-1LAI containing bulk-amplified, plasma-derived, full-length reverse transcriptase sequences. Fold change (FC) values were calculated compared with a composite 50% inhibitory concentration (IC50) from 12 recombinant subtype C HIV-1LAI plasma-derived viruses from treatment-naive individuals in South Africa. A total of 25/100 (25%) samples showed >500-FCs to DPV compared to treatment-naive samples with IC50s exceeding the maximum DPV concentration tested (132 ng/ml). A total of 66/100 (66%) samples displayed 3- to 306-FCs, with a median IC50 of 17.6 ng/ml. Only 9/100 (9%) samples were susceptible to DPV (FC < 3). Mutations L100I and K103N were significantly more frequent in samples with >500-fold resistance to DPV compared to samples with a ≤500-fold resistance. A total of 91% of samples with NNRTI-resistant HIV-1 from individuals on failing first-line ART in South Africa exhibited ≥3-fold cross-resistance to DPV. This level of resistance exceeds expected plasma concentrations, but very high genital tract DPV concentrations from DPV ring use could block viral replication. It is critically important to assess the frequency of transmitted and selected DPV resistance in individuals using the DPV ring.

Impact of SARS-CoV-2 Resistance to Antiviral Monoclonal Antibody Therapy on Neutralizing Antibody Response.

Background: Anti-SARS-CoV-2 monoclonal antibodies (mAbs) have played a key role as an anti-viral against SARS-CoV-2, but there is a potential for resistance to develop. The interplay between host antibody responses and the development of monoclonal antibody (mAb) resistance is a critical area of investigation. In this study, we assessed host neutralizing antibody (nAb) responses against both ancestral virus and those with treatment-emergent E484K bamlanivimab resistance mutations. Methods: Study participants were enrolled in the ACTIV-2/Advancing Clinical Therapeutics Globally (ACTG) A5401 phase 2 randomized, placebo-controlled trial of bamlanivimab 700 mg mAb therapy (NCT04518410). Anterior nasal and nasopharyngeal swabs were collected for SARS-CoV-2 RNA testing and S gene next-generation sequencing to identify the E484K bamlanivimab resistance mutation. Serum nAb titers were assessed by pseudovirus neutralization assays. Results: Higher baseline (pre-treatment) nAb titers against either ancestral or E484K virus was associated with lower baseline viral load. Participants with emerging resistance had low levels of nAb titers against either ancestral or E484K nAb at the time of study entry. Participants with emergent E484K resistance developed significantly higher levels of E484K-specific nAb titers compared to mAb-treated individuals who did not develop resistance. All participants who developed the E484K mAb resistance mutation were eventually able to clear the virus. Conclusion: Emerging drug resistance after SARS-CoV-2-specific mAb therapy led to a heightened host neutralizing antibody response to the mAb-resistant variant that was associated with eventual viral clearance. This demonstrates the interplay between the antiviral treatment-directed viral evolution and subsequent host immune response in viral clearance.

Comparison Study of the Bio-Plex and Meso Scale Multiplexed SARS-CoV-2 Serology Assays Reveals Evidence of Diminished Host Antibody Responses to SARS-CoV-2 after Monoclonal Antibody Treatment.

Background: Assessing the breadth and duration of antigen-specific binding antibodies provides valuable information for evaluating interventions to treat or prevent SARS-CoV-2 infection. Multiplex immunoassays are a convenient method for rapid measurement of antibody responses but can sometimes provide discordant results, and antibody positive percent agreement for COVID-19 diagnosis can vary depending on assay type, disease severity, and population sampled. Therefore, we compared two assays marked for research applications, MSD and Bio-Plex Pro, to evaluate qualitative interpretation of serostatus and quantitative detection of antibodies of varying isotypes (IgG, IgM, and IgA) against receptor binding domain (RBD) and nucleocapsid (N) antigens. Methods: Specimens from ACTIV-2/A5401, a placebo-controlled clinical trial of the SARSCoV-2 monoclonal antibody (mAb) bamlanivimab to prevent COVID-19 disease progression, were used to evaluate the concordance of the Bio-Rad Bio-Plex Pro Human SARS-CoV-2 Serology Assay and the Meso Scale Discovery (MSD) V-PLEX COVID-19 Panel 1 serology assay in detecting and quantifying IgG, IgA, and IgM binding anti-SARS-CoV-2 antibody responses against the RBD and N antigens. Data were disaggregated by study arm, bamlanivimab dose, days post-enrollment, and presence of emerging resistance. Results: We observed 90.5% (412 of 455 tests) concordance for anti-RBD IgG and 87% (396 of 455) concordance for anti-N IgG in classifying samples as negative or positive based on assay-defined cutoffs. Antibody levels converted to the WHO standard BAU/mL were significantly correlated for all isotypes (IgG, IgM, and IgA) and SARS-CoV-2 antigen targets (RBD and N) tested that were common between the two assays (Spearman r 0.65 to 0.92, P < 0.0001). Both assays uncovered evidence of diminished host-derived IgG immune responses in participants treated with bamlanivimab compared to placebo. Assessment of immune responses in the four individuals treated with the 700 mg of bamlanivimab with emerging mAb resistance demonstrated a stronger anti-N IgG response (MSD) at day 28 (median 2.18 log BAU/mL) compared to participants treated with bamlanivimab who did not develop resistance (median 1.55 log BAU/mL). Conclusions: These data demonstrate the utility in using multiplex immunoassays for characterizing the immune responses with and without treatment in a study population and provide evidence that monoclonal antibody treatment in acute COVID-19 may have a modest negative impact on development of host IgG responses.

Rapid determination of SARS-CoV-2 antibody neutralization titer using Bio-Rad Bio-Plex correlates strongly with pseudovirus-determined neutralization titer.

Accurate and rapid evaluation of SARS-CoV-2 half-maximal neutralizing antibody (nAb) titer (NT50) is an important research tool for measuring nAb responses after prophylaxis or therapeutics for COVID-19 prevention and management. Compared with ACE2-competitive enzyme immunoassays for nAb detection, pseudovirus assays remain low-throughput and labor intensive. A novel application of the Bio-Rad Bio-Plex Pro Human SARS-CoV-2 D614G S1 Variant nAb Assay was used to determine NT50 from COVID-19-vaccinated individuals and showed strong correlation to a laboratory-developed SARS-CoV-2 pseudovirus nAb assay. The Bio-Plex nAb assay could provide a rapid, high-throughput, culture-free method for NT50 determination in sera.

HIV-1 drug resistance among individuals who seroconverted in the ASPIRE dapivirine ring trial.

INTRODUCTION: A potential concern with the use of dapivirine (DPV) for HIV prevention is the selection of a drug-resistant virus that could spread and reduce the effectiveness of non-nucleoside reverse transcriptase (NNRTI)-based first-line antiretroviral therapy. We evaluated HIV-1 seroconversions in MTN-020/ASPIRE for selection of drug resistance and evaluated the genetic basis for observed reductions in susceptibility to DPV. METHODS: MTN-020/ASPIRE was a placebo-controlled, Phase III safety and effectiveness study of DPV ring for HIV-1 prevention conducted at 15 sites in South Africa, Zimbabwe, Malawi and Uganda between 2012 and 2015. Plasma from individuals who seroconverted in ASPIRE was analysed for HIV-1 drug resistance using both population Sanger sequencing and next-generation sequencing (NGS) with unique molecular identifiers to report mutations at ≥1% frequency. DPV susceptibility of plasma-derived recombinant HIV-1 containing bulk-cloned full-length reverse transcriptase sequences from MTN-020/ASPIRE seroconversions was determined in TZM-bl cells. Statistical significance was calculated using the Fisher's exact test. RESULTS: Plasma from all 168 HIV seroconversions were successfully tested by Sanger sequencing; 57 of 71 DPV arm and 82 of 97 placebo (PLB) arm participants had NGS results at 1% sensitivity. Overall, 18/168 (11%) had NNRTI mutations including K101E, K103N/S, V106M, V108I, E138A/G, V179D/I/T and H221Y. Five samples from both arms had low-frequency NNRTI mutations that were not detected by Sanger sequencing. The frequency of NNRTI mutations from the DPV arm (11%) was not different from the PLB arm (10%; p = 0.80). The E138A mutation was detected in both the DPV (3 of 71 [4.2%]) and PLB arm (5 of 97 [5.2%]) and conferred modest reductions in DPV susceptibility in some reverse transcriptase backgrounds but not others. CONCLUSIONS: HIV-1 drug resistance including NNRTI resistance did not differ between the DPV and placebo arms of the MTN-020/ASPIRE study, indicating that drug resistance was not preferentially acquired or selected by the DPV ring and that the preventive benefit of DPV ring outweighs resistance risk.

Frequent cross-resistance to rilpivirine among subtype C HIV-1 from first-line antiretroviral therapy failures in South Africa.

Background Rilpivirine (TMC278LA) is a promising drug for pre-exposure prophylaxis of HIV-1 because of its sub-nanomolar potency and long-acting formulation; however, increasing transmission of non-nucleoside reverse transcriptase inhibitor-resistant HIV-1 with potential cross-resistance to rilpivirine could reduce its preventive efficacy. This study investigated rilpivirine cross-resistance among recombinant subtype C HIV-1 derived from 100 individuals failing on first-line non-nucleoside reverse transcriptase inhibitor-containing antiretroviral therapy in South Africa whose samples were sent for routine HIV-1 drug resistance testing to Lancet Laboratories (Johannesburg, South Africa). Methods Plasma samples were selected from individuals with HIV-1 RNA > 10,000 copies/ml and ≥1 non-nucleoside reverse transcriptase inhibitor-resistance mutation in reverse transcriptase. Recombinant HIV-1LAI-containing bulk-cloned full-length reverse transcriptase sequences from plasma were assayed for susceptibility to nevirapine (NVP), efavirenz (EFV) and rilpivirine in TZM-bl cells. Fold-change (FC) decreases in drug susceptibility were calculated against a mean IC50 from 12 subtype C HIV-1 samples from treatment-naïve individuals in South Africa. Cross-resistance was evaluated based on biological cutoffs established for rilpivirine (2.5-FC) and the effect of mutation combinations on rilpivirine phenotype. Results Of the 100 samples from individuals on failing antiretroviral therapy, 69 had 2.5- to 75-fold decreased susceptibility to rilpivirine and 11 had >75-fold resistance. Rilpivirine resistance was strongly associated with K103N especially in combination with other rilpivirine-associated mutations. Conclusion The frequently observed cross-resistance of HIV-1 suggests that the preventive efficacy of TMC278LA pre-exposure prophylaxis could be compromised by transmission of HIV-1 from individuals with failure of first-line non-nucleoside reverse transcriptase inhibitor-containing antiretroviral therapy.

Susceptibility patterns of orally administered antimicrobials among urinary tract infection pathogens from hospitalized patients in North America: comparison report to Europe and Latin America. Results from the SENTRY Antimicrobial Surveillance Program (2000).

Urinary tract infections (UTIs) remain a worldwide nosocomial infection problem. Geographic variations in pathogen occurrence and susceptibility profiles require monitoring to provide information to guide new (garenoxacin [BMS284756]) therapeutic options. Two thousand seven hundred-eighty UTI isolates from Europe (n = 783), Latin America (531), and North America (1,466) were tested and compared against 44 agents by reference methods in the SENTRY Antimicrobial Surveillance Program. The top seven pathogens accounted for 90% of all isolates and the rank order for all regions was: Escherichia coli (1,316; 47%), Enterococcus spp. (351; 13%), Klebsiella spp. (306; 11%), Pseudomonas aeruginosa (210; 8%), Proteus mirabilis (145; 5%), Enterobacter spp. (97; 4%), and Citrobacter spp. (78; 3%). The pathogen rank order was similar among regions except for the rarer occurrence of Enterococcus spp. (Rank #6, 4%) in Latin America. E. coli ampicillin resistance was highest in Europe and Latin America (51-55%). Ampicillin (37%), ciprofloxacin or garenoxacin (4%), and trimethoprim/sulfamethoxazole (23%) resistance remained lowest in North America. Nitrofurantoin susceptibility in E. coli was still at acceptable levels and ranged from 91 to 96% across regions. The regional ciprofloxacin-resistant rank order for P. aeruginosa by region was: Latin America (55%) > Europe (41%) > North America (29%). Vancomycin-resistant enterococci (VRE) were only detected in North America (7%). Garenoxacin possessed a 34 to 44% wider spectrum compared to ciprofloxacin against enterococci UTI isolates. Extended spectrum beta-lactamase rates for E. coli and Klebsiella spp. were 4 and 19%, respectively. These results emphasized the need to assess the often striking differences in pathogen occurrence and resistance rates among the commonly encountered UTI pathogens.

Contemporary re-evaluation of the activity and spectrum of grepafloxacin tested against isolates in the United States.

Grepafloxacin potency and spectrum of activity were re-evaluated against contemporary pathogens collected from clinical infections in 2001-2002. A total of 995 isolates were tested for grepafloxacin by the reference agar dilution method and these results were compared to those of 25 other antimicrobial agents. Grepafloxacin activity remained comparable to that of ciprofloxacin, levofloxacin and gatifloxacin against Escherichia coli, Klebsiella pneumoniae and Enterobacter cloacae (MIC(90), 0.03-2 microg/ml; 0.0-7.7% resistance rates). For Pseudomonas aeruginosa, grepafloxacin was active against ciprofloxacin-susceptible (MIC(90), 2 microg/ml), but not against ciprofloxacin-resistant (MIC(90), >8 microg/ml) isolates. Against methicillin-susceptible Staphylococcus aureus, grepafloxacin susceptibility rate was 91.4%, equal to that of levofloxacin. None of the fluoroquinolones showed reasonable activity against methicillin-resistant staphylococci. Gatifloxacin and grepafloxacin had the same MIC(90) against beta-hemolytic streptococci (0.25 microg/ml) and penicillin-susceptible Streptococcus pneumoniae (0.25 microg/ml). Grepafloxacin and other fluoroquinolone activities were not influenced by penicillin resistance in S. pneumoniae. Grepafloxacin was very active against Haemophilus influenzae (MIC(90), 0.03 microg/ml), Moraxella catarrhalis (MIC(90), 0.03 microg/ml) and Legionella spp. (MIC(90), 0.5 microg/ml). These results on recently isolated organisms indicate that grepafloxacin has a sustained potency and spectrum against most clinically important and indicated pathogens.

Comparison of Streptococcus pneumoniae and Haemophilus influenzae susceptibilities from community-acquired respiratory tract infections and hospitalized patients with pneumonia: five-year results for the SENTRY Antimicrobial Surveillance Program.

The SENTRY Antimicrobial Surveillance Program has been monitoring the activity of commonly prescribed and novel antimicrobial agents on a global scale from 1997 to the present. Specific objectives have documented the key resistance rates among pathogens from both patients hospitalized with pneumonia and those diagnosed with community-acquired pneumonia. Hemophilus influenzae and Streptococcus pneumoniae are common pathogens in both of these patient populations and the susceptibility profiles for these two species were compared to distinguish potential differences that may be evident in North American surveillance (1997-2001). A total of 6,515 isolates of S. pneumoniae and 6,726 H. influenzae strains were tested using reference broth microdilution methods at a monitoring center. Ampicillin resistance was approximately 25% among H. influenzae isolates and did not significantly differ between strains from community-acquired infections or hospitalized patients. beta-lactamase-negative ampicillin resistant strains and fluoroquinolone refractory strains were rare (0.3 and

Antimicrobial susceptibility patterns of beta-hemolytic and viridans group streptococci: report from the SENTRY Antimicrobial Surveillance Program (1997-2000).

Susceptibility patterns of 15 antimicrobial agents were assessed for 3,400 isolates of beta-hemolytic (betahS) and viridans group (VgS) streptococci in the four regions of the SENTRY Antimicrobial Surveillance Program: Asia-Pacific (APAC), Europe (EU), Latin America (LA) and North America (NA). In 1997 through 2000, SENTRY Program monitors tested strains by reference broth microdilution methods and results were interpreted using National Committee for Clinical Laboratory Standards criteria. Among the betahS processed, 81.9% of strains were either Streptococcus pyogenes (n = 650) or S. agalactiae (n = 1,190). The VgS were generally classified as unspeciated alpha-hemolytic streptococci (n = 512; 44%) or S. mitis (n = 254; 22%). Seven quinolones, two beta-lactams, erythromycin (ER), clindamycin (CM), quinupristin/dalfopristin (Q/D), vancomycin (VA), teicoplanin (TP) and linezolid (LZ) were tested. Rank order of susceptibility for betahS isolates was: ceftriaxone (CTX) = Q/D = VA = TP = LZ (100.0%) > gatifloxacin (GATI) = trovafloxacin (TROV, 99.8%) > levofloxacin (LEVO; 99.7%) > penicillin (PEN; 99.3%) > grepafloxacin (GREPA; 97.4%) > CM (94.4%) > ER (85.5%). ER versus betahS had the highest MIC(90) values (2 microg/ml) and the lowest susceptibility rates across all regions (range, 81.4% in NA to 97.3% in LA). Among the VgS, susceptibility rank order was: VA = TP = LZ (100.0%) > Q/D (99.1%) > GATI = LEVO = TROVA (98.0%) > GREPA (96.5%) > CTX (92.8%) > CM (90.3%) > PEN (68.6%) > ER (64.5%). Macrolide resistance in both streptococcal species groups of the M-phenotype was highest in the Americas, with erm-patterns predominating in EU and APAC regions. BMS284756 among the monitored new agents showed a four- to eight-fold greater potency versus these streptococcal isolates when compared to the other six tested quinolones. Like Streptococcus pneumoniae, these other streptococci appear to have acquired numerous resistances and require continued surveillance to direct adequate therapies.

BMS284756 (formerly T-3811, a des-fluoroquinolone) potency and spectrum tested against over 10,000 bacterial bloodstream infection isolates from the SENTRY antimicrobial surveillance programme (2000).

BMS284756 is a novel des-F (6)-quinolone, which has a wide range of activity against many species of Gram-positive and -negative organisms. The potency of BMS284756 was compared with that of other quinolones, including ciprofloxacin, gatifloxacin and levofloxacin, and was tested against >10,000 bloodstream isolates from the year 2000 SENTRY antimicrobial surveillance programme. Twelve pathogens accounted for nearly all of the referred isolates and included Staphylococcus aureus, coagulase-negative staphylococci, Escherichia coli, Klebsiella pneumoniae, Enterobacter spp., Serratia spp., Pseudomonas aeruginosa, Acinetobacter spp., Enterococcus spp., Streptococcus pneumoniae and beta-haemolytic or viridans group streptococci. Of the four quinolones tested, BMS284756 was the most active overall against Staphylococcus spp. (MIC(50) < or = 0.03 mg/L) and Streptococcus spp. (MIC(50) 0.06 mg/L). In contrast, BMS284756 was less potent than the other quinolones against the enteric Gram-negative bacilli (MIC(50) < or = 0.03-1 mg/L). With a proposed breakpoint of < or =4 mg/L, BMS284756 may be a therapeutic alternative pending the results of clinical trials.

Commercial broth microdilution panel validation and reproducibility trials for garenoxacin (BMS-284756), a novel desfluoroquinolone.

Results from garenoxacin dry-form broth microdilution MIC panels prepared commercially (Sensititre, TREK Diagnostics) were compared to reference frozen-form MICs to ensure the validity of the longer-shelf-life product. A total of 1078 organisms from seven major organism groups were used in this trial. All commercial MIC results were within +/- one log(2) dilution of reference garenoxacin values, and reproducibility trials produced identical MIC results for 90.5 to 92.1% of garenoxacin MIC comparisons. Control quinolones (ciprofloxacin and gatifloxacin) also performed at a similarly high level of accuracy.