Chlamydial Pgp3 Seropositivity and Population-Attributable Fraction Among Women With Tubal Factor Infertility.

BACKGROUND: Chlamydial infection is associated with tubal factor infertility (TFI); however, assessment of prior chlamydial infection and TFI is imperfect. We previously evaluated a combination of serological assays for association with TFI. We now describe the chlamydial contribution to TFI using a newer Chlamydia trachomatis Pgp3-enhanced serological (Pgp3) assay. METHODS: In our case-control study of women 19 to 42 years old with hysterosalpingogram-diagnosed TFI (cases) and non-TFI (controls) in 2 US infertility clinics, we assessed possible associations and effect modifiers between Pgp3 seropositivity and TFI using adjusted odds ratios with 95% confidence intervals (CIs) stratified by race. We then estimated the adjusted chlamydia population-attributable fraction with 95% CI of TFI. RESULTS: All Black (n = 107) and 618 of 620 non-Black women had Pgp3 results. Pgp3 seropositivity was 25.9% (95% CI, 19.3%-33.8%) for non-Black cases, 15.2% (95% CI, 12.3%-18.7%) for non-Black controls, 66.0% (95% CI, 51.7%-77.8%) for Black cases, and 71.7% (95% CI, 59.2%-81.5%) for Black controls. Among 476 non-Black women without endometriosis (n = 476), Pgp3 was associated with TFI (adjusted odds ratio, 2.6 [95% CI, 1.5-4.4]), adjusting for clinic, age, and income; chlamydia TFI-adjusted population-attributable fraction was 19.8% (95% CI, 7.7%-32.2%) in these women. Pgp3 positivity was not associated with TFI among non-Black women with endometriosis or among Black women (regardless of endometriosis). CONCLUSIONS: Among non-Black infertile women without endometriosis in these clinics, 20% of TFI was attributed to chlamydia. Better biomarkers are needed to estimate chlamydia TFI PAF, especially in Black women.

Variability in detection of SARS-CoV-2-specific antibody responses following mild infection: a prospective multicentre cross-sectional study, London, United Kingdom, 17 April to 17 July 2020.

IntroductionImmunoassays targeting different SARS-CoV-2-specific antibodies are employed for seroprevalence studies. The degree of variability between immunoassays targeting anti-nucleocapsid (anti-NP; the majority) vs the potentially neutralising anti-spike antibodies (including anti-receptor-binding domain; anti-RBD), particularly in mild or asymptomatic disease, remains unclear.AimsWe aimed to explore variability in anti-NP and anti-RBD antibody detectability following mild symptomatic or asymptomatic SARS-CoV-2 infection and analyse antibody response for correlation with symptomatology.MethodsA multicentre prospective cross-sectional study was undertaken (April-July 2020). Paired serum samples were tested for anti-NP and anti-RBD IgG antibodies and reactivity expressed as binding ratios (BR). Multivariate linear regression was performed analysing age, sex, time since onset, symptomatology, anti-NP and anti-RBD antibody BR.ResultsWe included 906 adults. Antibody results (793/906; 87.5%; 95% confidence interval: 85.2-89.6) and BR strongly correlated (ρ = 0.75). PCR-confirmed cases were more frequently identified by anti-RBD (129/130) than anti-NP (123/130). Anti-RBD testing identified 83 of 325 (25.5%) cases otherwise reported as negative for anti-NP. Anti-NP presence (+1.75/unit increase; p < 0.001), fever (≥ 38°C; +1.81; p < 0.001) or anosmia (+1.91; p < 0.001) were significantly associated with increased anti-RBD BR. Age (p = 0.85), sex (p = 0.28) and cough (p = 0.35) were not. When time since symptom onset was considered, we did not observe a significant change in anti-RBD BR (p = 0.95) but did note decreasing anti-NP BR (p < 0.001).ConclusionSARS-CoV-2 anti-RBD IgG showed significant correlation with anti-NP IgG for absolute seroconversion and BR. Higher BR were seen in symptomatic individuals, particularly those with fever. Inter-assay variability (12.5%) was evident and raises considerations for optimising seroprevalence testing strategies/studies.

The Association Between Antibody Response to Severe Acute Respiratory Syndrome Coronavirus 2 Infection and Post-COVID-19 Syndrome in Healthcare Workers.

It is currently unknown how post-COVID-19 syndrome (PCS) may affect those infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This longitudinal study includes healthcare staff who tested positive for SARS-CoV-2 between March and April 2020, with follow-up of their antibody titers and symptoms. More than half (21 of 38) had PCS after 7-8 months. There was no statistically significant difference between initial reverse-transcription polymerase chain reaction titers or serial antibody levels between those who did and those who did not develop PCS. This study highlights the relative commonality of PCS in healthcare workers and this should be considered in vaccination scheduling and workforce planning to allow adequate frontline staffing numbers.

Asymptomatic Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection in a Rehabilitation Facility: Evolution of the Presence of Nasopharyngeal SARS-CoV-2 and Serological Antibody Responses.

At the start of the UK coronavirus disease 2019 epidemic, this rare point prevalence study revealed that one-third of patients (15 of 45) in a London inpatient rehabilitation unit were found to be infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) but asymptomatic. We report on 8 patients in detail, including their clinical stability, the evolution of their nasopharyngeal viral reverse-transcription polymerase chain reaction (RT-PCR) burden, and their antibody levels over time, revealing the infection dynamics by RT-PCR and serology during the acute phase. Notably, a novel serological test for antibodies against the receptor binding domain of SARS-CoV-2 showed that 100% of our asymptomatic cohort remained seropositive 3-6 weeks after diagnosis.

Longevity of SARS-CoV-2 immune responses in hemodialysis patients and protection against reinfection.

Patients with end stage kidney disease receiving in-center hemodialysis (ICHD) have had high rates of SARS-CoV-2 infection. Following infection, patients receiving ICHD frequently develop circulating antibodies to SARS-CoV-2, even with asymptomatic infection. Here, we investigated the durability and functionality of the immune responses to SARS-CoV-2 infection in patients receiving ICHD. Three hundred and fifty-six such patients were longitudinally screened for SARS-CoV-2 antibodies and underwent routine PCR-testing for symptomatic and asymptomatic infection. Patients were regularly screened for nucleocapsid protein (anti-NP) and receptor binding domain (anti-RBD) antibodies, and those who became seronegative at six months were screened for SARS-CoV-2 specific T-cell responses. One hundred and twenty-nine (36.2%) patients had detectable antibody to anti-NP at time zero, of whom 127 also had detectable anti-RBD. Significantly, at six months, 71/111 (64.0%) and 99/116 (85.3%) remained anti-NP and anti-RBD seropositive, respectively. For patients who retained antibody, both anti-NP and anti-RBD levels were reduced significantly after six months. Eleven patients who were anti-NP seropositive at time zero, had no detectable antibody at six months; of whom eight were found to have SARS-CoV-2 antigen specific T cell responses. Independent of antibody status at six months, patients with baseline positive SARS-CoV-2 serology were significantly less likely to have PCR confirmed infection over the following six months. Thus, patients receiving ICHD mount durable immune responses six months post SARS-CoV-2 infection, with fewer than 3% of patients showing no evidence of humoral or cellular immunity.

Severe Acute Respiratory Syndrome Coronavirus-2 Infections in Critical Care Staff: Beware the Risks Beyond the Bedside.

OBJECTIVES: Critical care workers were considered to be at high risk of severe acute respiratory syndrome coronavirus-2 infection from patients during the first wave of the pandemic. Staff symptoms, previous swab testing, and antibody prevalence were correlated with patient admissions to investigate this assumption. DESIGN: Cross-sectional study. SETTING: A large critical care department in a tertiary-care teaching hospital in London, United Kingdom. SUBJECTS: Staff working in critical care. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Participants completed a questionnaire and provided a serum sample for severe acute respiratory syndrome coronavirus-2 antibody testing over a 3-day period in April 2020. We compared the timing of symptoms in staff to the coronavirus disease 2019 patient admissions to critical care. We also identified factors associated with antibody detection. Of 625 staff 384 (61.4%) reported previous symptoms and 124 (19.8%) had sent a swab for testing. Severe acute respiratory syndrome coronavirus-2 infection had been confirmed in 37 of those swabbed (29.8%). Overall, 21% (131/625) had detectable severe acute respiratory syndrome coronavirus-2 antibody, of whom 9.9% (13/131) had been asymptomatic. The peak onset of symptoms among staff occurred 2 weeks before the peak in coronavirus disease 2019 patient admissions. Staff who worked in multiple departments across the hospital were more likely to be seropositive. Staff with a symptomatic household contact were also more likely to be seropositive at 31.3%, compared with 16.2% in those without (p < 0.0001). CONCLUSIONS: Staff who developed coronavirus disease 2019 were less likely to have caught it from their patients in critical care. Other staff, other areas of the hospital, and the wider community are more likely sources of infection. These findings indicate that personal protective equipment was effective at preventing transmission from patients. However, staff also need to maintain protective measures away from the bedside.

Clinical and laboratory evaluation of SARS-CoV-2 lateral flow assays for use in a national COVID-19 seroprevalence survey.

BACKGROUND: Accurate antibody tests are essential to monitor the SARS-CoV-2 pandemic. Lateral flow immunoassays (LFIAs) can deliver testing at scale. However, reported performance varies, and sensitivity analyses have generally been conducted on serum from hospitalised patients. For use in community testing, evaluation of finger-prick self-tests, in non-hospitalised individuals, is required. METHODS: Sensitivity analysis was conducted on 276 non-hospitalised participants. All had tested positive for SARS-CoV-2 by reverse transcription PCR and were ≥21 days from symptom onset. In phase I, we evaluated five LFIAs in clinic (with finger prick) and laboratory (with blood and sera) in comparison to (1) PCR-confirmed infection and (2) presence of SARS-CoV-2 antibodies on two 'in-house' ELISAs. Specificity analysis was performed on 500 prepandemic sera. In phase II, six additional LFIAs were assessed with serum. FINDINGS: 95% (95% CI 92.2% to 97.3%) of the infected cohort had detectable antibodies on at least one ELISA. LFIA sensitivity was variable, but significantly inferior to ELISA in 8 out of 11 assessed. Of LFIAs assessed in both clinic and laboratory, finger-prick self-test sensitivity varied from 21% to 92% versus PCR-confirmed cases and from 22% to 96% versus composite ELISA positives. Concordance between finger-prick and serum testing was at best moderate (kappa 0.56) and, at worst, slight (kappa 0.13). All LFIAs had high specificity (97.2%-99.8%). INTERPRETATION: LFIA sensitivity and sample concordance is variable, highlighting the importance of evaluations in setting of intended use. This rigorous approach to LFIA evaluation identified a test with high specificity (98.6% (95%CI 97.1% to 99.4%)), moderate sensitivity (84.4% with finger prick (95% CI 70.5% to 93.5%)) and moderate concordance, suitable for seroprevalence surveys.

Genetic influence of ABCG2, UGT1A1 and NR1I2 on dolutegravir plasma pharmacokinetics.

OBJECTIVES: Dolutegravir has replaced efavirenz as first-line treatment in universal HIV guidelines. We sought to ascertain the contributory effect of SNPs in four key genes linked to dolutegravir disposition (UGT1A1, ABCG2, CYP3A and NR1I2) on plasma dolutegravir pharmacokinetics. METHODS: Paired pharmacogenetic/pharmacokinetic data from 93 subjects were analysed for association using multivariate linear regression. RESULTS: Co-occurring UGT1*28 and NR1I2 c.63396C>T homozygosity was associated with a 79% increase in AUC0-24 (P = 0.001; 27% if analysed individually), whilst combined ABCG2 c.421C>A and NR1I2 c.63396C>T variants were associated with a 43% increase in Cmax (P = 0.002) and a 39% increase in AUC0-24 (P = 0.002). When analysed individually, homozygosity for the NR1I2 c.63396C>T variant alleles was associated with a 28% increase in Cmax (P = 0.033) and homozygosity for the ABCG2 c.421C>A variant alleles was associated with a 28% increase in Cmax (P = 0.047). The UGT1A1*28 (rs8175347) poor metabolizer status (*28/*28; *28/*37; *37/*37) was individually associated with a 27% increase in AUC0-24 (P = 0.020). The combination of UGT1A1*28 poor metabolizer and UGT1A1*6 intermediate metabolizer statuses correlated with a 43% increase in AUC0-24 (P = 0.023). CONCLUSIONS: This study showed a pharmacogenetic association between dolutegravir pharmacokinetics and variants in the ABCG2, UGT1A1 and NR1I2 genes, particularly when combined. Further research is warranted to confirm these associations in population-specific studies and to investigate their putative relationship with dolutegravir pharmacodynamics.

Pharmacokinetics of Efavirenz 400 mg Once Daily Coadministered With Isoniazid and Rifampicin in Human Immunodeficiency Virus-Infected Individuals.

Background: The World Health Organization recommends efavirenz 400 mg (EFV400) as first-line antiretroviral therapy, with a disclaimer that no data with anti-tuberculosis (TB) treatment exist. Many people living with human immunodeficiency virus (PLWH) require TB treatment with isoniazid (INH) and rifampicin (RIF), which affect cytochrome P450 and antiretroviral exposure. Methods: PLWH receiving tenofovir disoproxil fumarate (TDF)/emtricitabine (FTC)/EFV 600 mg with a viral load (VL) <50 copies/mL switched to TDF/FTC/EFV400. Genetic polymorphisms and pharmacokinetic (PK) parameters of EFV400 without (PK1) and with INH/RIF following 4 (PK2) and 12 (PK3) weeks of coadministration were evaluated. Results: Twenty-six PLWH were enrolled; 22 completed PK2. All maintained VL <50 copies/mL throughout the study. Geometric mean ratio (GMR) PK2/PK1 of EFV400 maximum plasma concentration (Cmax), area under the curve (AUC), and concentration at 24 hours postdose (C24h) were 0.91 (90% confidence interval [CI], .83-.99), 0.91 (90% CI, .79-1.05), and 0.85 (90% CI, .72-.99), respectively. GMRs (90% CI) of PK3/PK2 and PK3/PK1 Cmax, AUC, and C24h were 0.95 (.86-1.05) and 0.92 (.83-1.01), 0.88 (.75-1.03) and 0.84 (.75-.93), and 0.84 (.72-.99) and 0.75 (.62-.92), respectively. Eleven of 22 participants carried polymorphisms in the CYP2B6 gene associated with slow EFV metabolism. Conclusions: INH/RIF coadministration was associated with limited changes in EFV400 AUC (<25%), and EFV400 concentrations were maintained within ranges of those measured in PLWH in the ENCORE-1 study, irrespective of CYP2B6 genotype. The coadministration of EFV400 with anti-TB treatment can be considered and this is being confirmed in PLWH with TB. Clinical Trials Registration: NCT02832778.

Increased Dolutegravir Peak Concentrations in People Living With Human Immunodeficiency Virus Aged 60 and Over, and Analysis of Sleep Quality and Cognition.

Background: Demographic data show an increasingly aging human immunodeficiency virus (HIV) population worldwide. Recent concerns over dolutegravir-related neuropsychiatric toxicity have emerged, particularly amongst older people living with HIV (PLWH). We describe the pharmacokinetics (PK) of dolutegravir (DTG) 50 mg once daily in PLWH aged 60 and older. Additionally, to address calls for prospective neuropsychiatric toxicodynamic data, we evaluated changes in sleep quality and cognitive functioning in this population after switching to abacavir (ABC)/lamivudine (3TC)/DTG over 6 months. Methods: PLWH ≥60 years with HIV-viral load <50 copies/mL on any non-DTG-based antiretroviral combination were switched to ABC/3TC/DTG. On day 28, 24-hour PK sampling was undertaken. Steady-state PK parameters were compared to a published historical control population aged ≤50 years. We administered 6 validated sleep questionnaires and neurocognitive (Cogstate) testing pre-switch and over 180 days. Results: In total, 43 participants enrolled, and 40 completed the PK phase. Overall, 5 discontinued (2 due to sleep-related adverse events, 4.6%). DTG maximum concentration (Cmax) was significantly higher in patients ≥60 years old versus controls (geometric mean 4246 ng/mL versus 3402 ng/mL, P = .005). In those who completed day 180 (n = 38), sleep impairment (Pittsburgh Sleep Quality Index) was marginally higher at day 28 (P = .02), but not at days 90 or 180. Insomnia, daytime functioning, and fatigue test scores did not change statistically over time. Conclusions: DTG Cmax was significantly higher in older PLWH. Our data provides clinicians with key information on the safety of prescribing DTG in older PLWH.

Pharmacokinetics, Pharmacodynamics, and Pharmacogenetics of Efavirenz 400 mg Once Daily During Pregnancy and Post-Partum.

Background: A clinical trial showed that efavirenz 400 mg once daily (EFV400) is as effective as the standard adult dose. World Health Organization recommends EFV400 as an alternative first-line agent, but data are lacking in the third trimester of pregnancy (TT). We investigated the pharmacokinetics, efficacy, and CYP2B6 pharmacogenetics in HIV-infected women (WLWH) on EFV400 during TT and post-partum (PP). Methods: An open-label 2-center study (United Kingdom, Uganda) was conducted in WLWH receiving antiretroviral regimens containing efavirenz 600 mg, who had their efavirenz dose reduced to EFV400. Weekly therapeutic drug monitoring (TDM), steady-state pharmacokinetic profiles (TT and PP), safety, virological efficacy, and CYP2B6 polymorphisms at positions 516 (C > T) and 938 (T > C) were evaluated. Results: Twenty-five WLWH of African origin were enrolled. All had viral loads <50 copies/mL at baseline, which were maintained throughout the study. No infant was HIV infected. No WLWH were withdrawn due to low EFV400 TDM results. Geometric mean ratios (TT/PP; 90% confidence interval) for EFV400 maximum observed plasma concentration, area under the curve, and plasma concentration measured 24 hours after the observed dose were 0.97 (.85-1.10), 0.87 (.76-.99), and 0.77 (.65-.91), respectively. Five of 25 WLWH were slow metabolizers. Conclusions: Although EFV400 pharmacokinetic parameters were slightly lower for TT compared with PP values, efavirenz concentrations exceeded cutoff levels established by the study and those measured in antiretroviral-naive patients receiving EFV400 in ENCORE1. All subjects maintained a viral load <50 copies/mL, suggesting that EFV400 can be used in pregnant WLWH.

The Rsb Phosphoregulatory Network Controls Availability of the Primary Sigma Factor in Chlamydia trachomatis and Influences the Kinetics of Growth and Development.

Chlamydia trachomatis is an obligate intracellular human pathogen that exhibits stage-specific gene transcription throughout a biphasic developmental cycle. The mechanisms that control modulation in transcription and associated phenotypic changes are poorly understood. This study provides evidence that a switch-protein kinase regulatory network controls availability of σ66, the main sigma subunit for transcription in Chlamydia. In vitro analysis revealed that a putative switch-protein kinase regulator, RsbW, is capable of interacting directly with σ66, as well as phosphorylating its own antagonist, RsbV1, rendering it inactive. Conversely, the putative PP2C-like phosphatase domain of chlamydial RsbU was capable of reverting RsbV1 into its active state. Recent advances in genetic manipulation of Chlamydia were employed to inactivate rsbV1, as well as to increase the expression levels of rsbW or rsbV1, in vivo. Representative σ66-dependent gene transcription was repressed in the absence of rsbV1 or upon increased expression of RsbW, and increased upon elevated expression of RsbV1. These effects on housekeeping transcription were also correlated to several measures of growth and development. A model is proposed where the relative levels of active antagonist (RsbV1) and switch-protein anti-sigma factor (RsbW) control the availability of σ66 and subsequently act as a molecular 'throttle' for Chlamydia growth and development.

Chlamydia trachomatis Incidence Using Self-Reports and Serology by Gender, Age Period, and Sexual Behavior in a Birth Cohort.

BACKGROUND: Although understanding chlamydia incidence assists prevention and control, analyses based on diagnosed infections may distort the findings. Therefore, we determined incidence and examined risks in a birth cohort based on self-reports and serology. METHODS: Self-reported chlamydia and behavior data were collected from a cohort born in New Zealand in 1972/3 on several occasions to age 38 years. Sera drawn at ages 26, 32, and 38 years were tested for antibodies to Chlamydia trachomatis Pgp3 antigen using a recently developed assay, more sensitive in women (82.9%) than men (54.4%). Chlamydia incidence by age period (first coitus to age 26, 26-32, and 32-38 years) was calculated combining self-reports and serostatus and risk factors investigated by Poisson regression. RESULTS: By age 38 years, 32.7% of women and 20.9% of men had seroconverted or self-reported a diagnosis. The highest incidence rate was to age 26, 32.7 and 18.4 years per 1000 person-years for women and men, respectively. Incidence rates increased substantially with increasing number of sexual partners. After adjusting age period incidence rates for partner numbers, a relationship with age was not detected until 32 to 38 years, and then only for women. CONCLUSIONS: Chlamydia was common in this cohort by age 38, despite the moderate incidence rates by age period. The strongest risk factor for incident infection was the number of sexual partners. Age, up to 32 years, was not an independent factor after accounting for partner numbers, and then only for women. Behavior is more important than age when considering prevention strategies.

Rapid and Efficient Stable Gene Transfer to Mesenchymal Stromal Cells Using a Modified Foamy Virus Vector.

Mesenchymal stromal cells (MSCs) hold great promise for regenerative medicine. Stable ex vivo gene transfer to MSCs could improve the outcome and scope of MSC therapy, but current vectors require multiple rounds of transduction, involve genotoxic viral promoters and/or the addition of cytotoxic cationic polymers in order to achieve efficient transduction. We describe a self-inactivating foamy virus vector (FVV), incorporating the simian macaque foamy virus envelope and using physiological promoters, which efficiently transduces murine MSCs (mMSCs) in a single-round. High and sustained expression of the transgene, whether GFP or the lysosomal enzyme, arylsulphatase A (ARSA), was achieved. Defining MSC characteristics (surface marker expression and differentiation potential), as well as long-term engraftment and distribution in the murine brain following intracerebroventricular delivery, are unaffected by FVV transduction. Similarly, greater than 95% of human MSCs (hMSCs) were stably transduced using the same vector, facilitating human application. This work describes the best stable gene transfer vector available for mMSCs and hMSCs.

Short-course antiretroviral therapy in primary HIV infection.

BACKGROUND: Short-course antiretroviral therapy (ART) in primary human immunodeficiency virus (HIV) infection may delay disease progression but has not been adequately evaluated. METHODS: We randomly assigned adults with primary HIV infection to ART for 48 weeks, ART for 12 weeks, or no ART (standard of care), with treatment initiated within 6 months after seroconversion. The primary end point was a CD4+ count of less than 350 cells per cubic millimeter or long-term ART initiation. RESULTS: A total of 366 participants (60% men) underwent randomization to 48-week ART (123 participants), 12-week ART (120), or standard care (123), with an average follow-up of 4.2 years. The primary end point was reached in 50% of the 48-week ART group, as compared with 61% in each of the 12-week ART and standard-care groups. The average hazard ratio was 0.63 (95% confidence interval [CI], 0.45 to 0.90; P=0.01) for 48-week ART as compared with standard care and was 0.93 (95% CI, 0.67 to 1.29; P=0.67) for 12-week ART as compared with standard care. The proportion of participants who had a CD4+ count of less than 350 cells per cubic millimeter was 28% in the 48-week ART group, 40% in the 12-week group, and 40% in the standard-care group. Corresponding values for long-term ART initiation were 22%, 21%, and 22%. The median time to the primary end point was 65 weeks (95% CI, 17 to 114) longer with 48-week ART than with standard care. Post hoc analysis identified a trend toward a greater interval between ART initiation and the primary end point the closer that ART was initiated to estimated seroconversion (P=0.09), and 48-week ART conferred a reduction in the HIV RNA level of 0.44 log(10) copies per milliliter (95% CI, 0.25 to 0.64) 36 weeks after the completion of short-course therapy. There were no significant between-group differences in the incidence of the acquired immunodeficiency syndrome, death, or serious adverse events. CONCLUSIONS: A 48-week course of ART in patients with primary HIV infection delayed disease progression, although not significantly longer than the duration of the treatment. There was no evidence of adverse effects of ART interruption on the clinical outcome. (Funded by the Wellcome Trust; SPARTAC Controlled-Trials.com number, ISRCTN76742797, and EudraCT number, 2004-000446-20.).

Determination of Sequences Required for Human Endogenous Retrovirus K Transduction and Its Recognition by Foreign Retroviral Virions.

Sequences necessary for transduction of human endogenous retrovirus (HERV)-Kcon, a consensus of the HERV-K(HML-2) family, were analyzed and found to reside in the leader/gag region. They act in an orientation-dependent way and consist of at least two sites working together. Having defined these sequences, we exploited this information to produce a simple system to investigate to what extent virions of HERV-Kcon, murine leukemia virus, and HIV-1 have the ability to transduce each other's genomes, leading to potential contamination of gene therapy vectors.

A Validated Method for Quantification of Dolutegravir Using Ultra Performance Liquid Chromatography Coupled With UV Detection.

BACKGROUND: Dolutegravir (DTG) is an integrase strand transfer inhibitor, which is a newly approved antiretroviral drug used for the treatment of HIV-infected naive and experienced individuals. Many aspects of DTG pharmacology remain to be studied. Our aim was to develop and fully validate a robust analytical method for the quantification of DTG in plasma using liquid chromatography coupled with UV detection. METHODS: A simple and rapid protein precipitation method was used for analyte extraction from 100 µL plasma. The separation was achieved on a C8 reverse-phase analytical column using a gradient elution with 50 mmol/L formic acid and 50 mmol/L ammonium acetate in water (mobile phase A), and 100% acetonitrile (mobile phase B) and at a flow rate of 0.3 mL/min and a total run time of 10 minutes. The detector wavelength was set at 258 nm. RESULTS: The linearity of the calibration curve (r > 0.9999, n = 6) was validated over a concentration range of 0.25-10 mcg/mL. Intra-assay variability ranged from 3.3% to 6.1% and inter-assay variability ranged from 4.5% to 5.7%. The overall accuracy ranged from 90.7% to 97.7% for the 3 different concentrations of quality control samples. Recovery efficiency of extraction ranged from 94.3%-100%. This method is highly selective with no interferences from commonly concomitant antiretroviral drugs or endogenous metabolites. CONCLUSIONS: The described method is simple, robust, selective, accurate, precise, and cost-effective. Thus, this assay can be readily transferred and implemented in clinical settings and used for pharmacokinetic studies and therapeutic drug monitoring programs.