Mass Spectrometry to Determine Intracellular Concentrations of Antiretroviral Drugs: From chemistry to clinical application

Around 1995 – 1996, treatment options for patients infected with the human immunodefiency virus (HIV), the causative agent of acquired immunodeficiency syndrome (AIDS) 1, 2, improved dramatically. Therapy with a combination of several classes of antiretroviral

Viruses as sole causative agents of severe acute respiratory tract infections in children

Background: Respiratory syncytial virus (RSV) and influenza A viruses are known to cause severe acute respiratory tract infections (SARIs) in children. For other viruses like human rhinoviruses (HRVs) this is less well established. Viral or bacterial co-infections are often considered essential for severe manifestations of these virus infections. Objective: The study aims at identifying viruses that may cause SARI in children in the absence of viral and bacterial co-infections, at identifying disease characteristics associated with these single virus infections, and at identifying a possible correlation between viral loads and disease severities. Study Design: Between April 2007 and March 2012, we identified children (<18 year) with or without a medical history, admitted to our paediatric intensive care unit (PICU) with SARI or to the medium care (MC) with an acute respiratory tract infection (ARTI) (controls). Data were extracted from the clinical and laboratory databases of our tertiary care paediatric hospital. Patient specimens were tested for fifteen respiratory viruses with real-time reverse transcriptase PCR assays and we selected patients with a single virus infection only. Typical bacterial co-infections were considered unlikely to have contributed to the PICU or MC admission based on C-reactive protein-levels or bacteriological test results if performed. Results: We identified 44 patients admitted to PICU with SARI and 40 patients admitted to MC with ARTI. Twelve viruses were associated with SARI, ten of which were also associated with ARTI in the absence of typical bacterial and viral co-infections, with RSV and HRV being the most frequent causes. Viral loads were not different between PICU-SARI patients and MCARTI patients. Conclusion: B

Prevalence of intrathecal acyclovir resistant virus in herpes simplex encephalitis patients

Herpes simplex encephalitis (HSE) is a life-threatening complication of herpes simplex virus (HSV) infection. Acyclovir (ACV) is the antiviral treatment of choice, but may lead to emergence of ACV-resistant (ACVR) HSV due to mutations in the viral UL23 gene encoding for the ACV-targeted thymidine kinase (TK) protein. Here, we determined the prevalence of intrathecal ACVR-associated HSV TK mutations in HSE patients and compared TK genotypes of sequential HSV isolates in paired cerebrospinal fluid (CSF) and blister fluid of mucosal HSV lesions. Clinical samples were obtained from 12 HSE patients, encompassing 4 HSV type 1 (HSV-1) and 8 HSV-2 encephalitis patients. HSV DNA load was determined by real-time PCR and complete HSV TK gene sequences were obtained by nested PCR followed by Sanger sequencing. All HSV-1 HSE patients contained viral TK mutations encompassing 30 unique nucleotide and 13 distinct amino acid mutations. By contrast, a total of 5 unique nucleotide and 4 distinct amino acid changes were detected in 7 of 8 HSV-2 patients. Detected mutations were identified as natural polymorphisms located in non-conserved HSV TK gene regions. ACV therapy did not induce the emergence of ACVR-associated HSV TK mutations in consecutive CSF and mucocutaneous samples of 5 individual patients. Phenotypic susceptibility analysis of these mucocutaneous HSV isolates demonstrated ACV-sensitive virus in 2 HSV-1 HSE patients, whereas in two HSV-2 HSE patients ACVR virus was detected in the absence of known ACVR-associated TK mutations. In conclusion, we did not detect intrathecal ACVR-associated TK mutations in HSV isolates obtained from 12 HSE patients

Dried blood spot UHPLC-MS/MS analysis of oseltamivir and oseltamivircarboxylate-a validated assay for the clinic

The neuraminidase inhibitor oseltamivir (Tamiflu®) is currently the first-line therapy for patients with influenza virus infection. Common analysis of the prodrug and its active metabolite oseltamivircarboxylate is determined via extraction from plasma. Compared with these assays, dried blood spot (DBS) analysis provides several advantages, including a minimum sample volume required for the measurement of drugs in whole blood. Samples can easily be obtained via a simple, non-invasive finger or heel prick. Mainly, these characteristics make DBS an ideal tool for pediatrics and to measure multiple time points such as those needed in therapeutic drug monitoring or pharmacokinetic studies. Additionally, DBS sample preparation, stability, and storage are usually most convenient. In the present work, we developed and fully validated a DBS assay for the simultaneous determination of oseltamivir and oseltamivircarboxylate concentrations in human whole blood. We demonstrate the simplicity of DBS sample preparation, and a fast, accurate and reproducible analysis using ultra high-performance liquid chromatography coupled to a triple quadrupole mass spectrometer. A thorough validation on the basis of the most recent FDA guidelines for bioanalytical method validation showed that the method is selective, precise, and accurate (≤15% RSD), and sensitive over the relevant clinical range of 5-1,500 ng/mL for oseltamivir and 20-1,500 ng/mL for the oseltamivircarboxyl

Targeted HCV core antigen monitoring among HIV-positive men who have sex with men is cost-saving

Introduction: The World Health Organization declared the goal of hepatitis C virus (HCV) elimination by 2030. Microelimination, which is the reduction of incidence to zero in targeted populations, is less complex and costly and may be the first step to prove whether elimination is feasible. A suitable target group are HIV-positive men who have sex with men (MSM) because of their high-risk behaviour and high incidence rates. Moreover, HCV monitoring is integrated in HIV care. The current HCV monitoring approach is suboptimal and complex and may miss new HCV infections. Alternative monitoring strategies, based on alanine aminotransferase, HCV-PCR and HCV-core antigen (HCV-cAg), combined with immediate direct-acting antiviral (DAA) treatment, may be more effective in reducing new HCV infections. Methods: A deterministic mathematical transmission model was constructed representing the Dutch HCV epidemic among HIV-positive MSM to compare different HCV monitoring strategies from 2018 onwards. We evaluated the epidemiological impact of alternative and intensified monitoring in MSM with HCV. In addition, the cost-effectiveness was calculated over a lifetime horizon. Results: Current HCV monitoring and treatment is projected to result in an incidence of 1.1/1000 person-years, 0.24% prevalence, at a cost of €61.8 million (interquartile range 52.2–73.9). Compared with current monitoring, intensified monitoring will result in a maximum 27% reduction of incidence and 33% in prevalence at an increased cost. Conversely, compared with current monitoring, targeted HCV-cAg monitoring will result in a comparable incidence (1.1/1000 person-years) and prevalence (0.23%) but will be €1 million cheaper with increased quality-adjusted life year. Conclusion: Targeted monitoring reduces the HCV epidemic in a cost-saving manner; however, micro-elimination may not be obtained by 2030, highlighting the need for harm-reduction programmes

Development of C-TILDA: A modified TILDA method for reservoir quantification in long term treated patients infected with subtype C HIV-1

A better characterization of the HIV reservoir is pivotal for the development of effective eradication strategies. Accurate quantification of the latent reservoir remains challenging. Starting from a regular blood draw, the Tat/Rev induced limiting dilution assay (TILDA) combines serial dilution of CD4+ T cells with a PCR-based detection of HIV-1 spliced mRNA produced upon cell stimulation. Here we adapted the original protocol for HIV-1 subtype B to detect tat/rev mRNAs transcribed from reactivated latently infected cells in long term suppressed patients infected with HIV-1 subtype C. Given the heterogeneity of global HIV epidemiology, it is pivotal to develop assays with optimal performances also in patients infected with non-B subtypes. We observed that, in these patients infected with subtype C virus, the HIV reservoir quantified by TILDA correlates with both the time of virological suppression and CD4/CD8 ratio

Ferrets as a novel animal model for studying human respiratory syncytial virus infections in immunocompetent and immunocompromised hosts

Human respiratory syncytial virus (HRSV) is an important cause of severe respiratory tract disease in immunocompromised patients. Animal models are indispensable for evaluating novel intervention strategies in this complex patient population. To complement existing models in rodents and non-human primates, we have evaluated the potential benefits of an HRSV infection model in ferrets (Mustela putorius furo). Nine- to 12-month-old HRSV-seronegative immunocompetent or immunocompromised ferrets were infected with a low-passage wild-type strain of HRSV subgroup A (105 TCID50) administered by intra-tracheal or intra-nasal inoculation. Immune suppression was achieved by bi-daily oral administration of tacrolimus, mycophenolate mofetil, and prednisolone. Throat and nose swabs were collected daily and animals were euthanized four, seven, or 21 days post-infection (DPI). Virus loads were determined by quantitative virus culture and qPCR. We observed efficient HRSV replication in both the upper and lower respiratory tract. In immunocompromised ferrets, virus loads reached higher levels and showed delayed clearance as compared to those in immunocompetent animals. Histopathological evaluation of animals euthanized 4 DPI demonstrated that the virus replicated in the respiratory epithelial cells of the trachea, bronchi, and bronchioles. These animal models can contribute to an assessment of the efficacy and safety of novel HRSV intervention strategies

The Association Between Cytomegalovirus Infection and Cardiac Allograft Vasculopathy in the Era of Antiviral Valganciclovir Prophylaxis

Background. Previous studies on the association between cytomegalovirus (CMV) infection and cardiac allograft vasculopathy (CAV) were conducted on patients transplanted in the prevalganciclovir prophylaxis era. The aim of our study is to evaluate this relation in heart transplantation (HTx) recipients treated according to current prophylactic and immunosuppressive regimens. Methods. This single-center retrospective study included all consecutive adult patients that underwent HTx between January 1, 2000, and May 31, 2018. Clinically relevant CMV infection was defined as either plasma CMV DNAemia ≥ 1000 IU/mL with/without clinical symptoms or <1000 IU/mL with symptoms. The primary endpoint was first manifestation of CAV diagnosed by coronary angiography. For statistical analysis, the cause-specific hazard regression model was applied, with clinically relevant CMV infection and any CMV infection as time-dependent variables. Results. In total, 260 patients were included in the analysis. The median (interquartile range) follow-up was 7.88 (4.21–12.04) years. During the follow-up, clinically relevant CMV infection was diagnosed in 96 (37%) patients and CAV in 149 (57%) patients. In the multivariate regression analysis, independent predictors of CAV were: number of rejection episodes (cause-specific hazard ratio [95% confidence interval]: 1.18 [1.04-1.34], P = 0.01), hypertension (1.61 [1.11-2.34], P = 0.01), treatment with mycophenolate mofetil (0.68 [0.47-0.97], P = 0.03). No significant association was observed between CMV infection and CAV, except for patients who experienced a breakthrough CMV infection (n = 24) during prophylaxis (1.94 [1.11-3.40], P = 0.02). Conclusions. In the era of contemporary immunosuppression and valganciclovir prophylaxis, a signifi

Emergence and Persistence of Letermovir-Resistant Cytomegalovirus in a Patient with Primary Immunodeficiency

Background: Letermovir is a novel cytomegalovirus antiviral that is approved for prophylaxis in hematopoietic stem cell transplantation recipients Methods: After obtaining informed consent, letermovir prophylaxis was started in a patient with a presumed late-onset primary, combined T-and B-cell immunodeficiency. Plasma CMV DNAemia was monitored with real-time polymerase chain reaction, and letermovir resistance analyses were performed using Sanger sequencing and Illumina MiSeq next-generation sequencing. Results: A letermovir-resistant cytomegalovirus variant (C325Y mutation in UL56) emerged 17 weeks after start of prophylaxis. The letermovir-resistant variant was able to reactivate without drug selective pressure as this variant was again detected in plasma 20.6 weeks after stopping of letermovir. Conclusions: This case indicates that the C325Y mutation in UL56 does not significantly alter fitness of cytomegalovirus in vivo

Predictors of virological failure in HIV-1-infected patients switching to dolutegravir maintenance monotherapy

Objectives: The Dolutegravir Monotherapy for HIV (DOMONO; NCT02401828) study showed that maintenance monotherapy with dolutegravir (DTG) is associated with virological failure (VF) and leads to DTG resistance and as a result should not be used. However, data on clinical and virological factors associated with VF during DTG monotherapy are lacking. We identified factors associated with VF during DTG monotherapy. Methods: A randomized trial was carried out in which patients on combination antiretroviral therapy (cART) with an HIV-1 RNA zenith < 100 000 copies/mL and a CD4 T-cell nadir ≥ 200 cells/μL, who had never experienced VF, switched to DTG monotherapy. Clinical and virological factors were compared between patients with and without VF, using univariate analyses. Results: Eight of the 95 patients developed VF during DTG monotherapy. A total of 78 participants had reached week 48 when the study was discontinued. The median CD4 T-cell nadir was lower in patients with VF than in patients without VF [260 (interquartile range (IQR) 223–320) versus 380 (IQR 290–520) cells/μL, respectively; P = 0.011]. Patients with VF had a longer time between HIV diagnosis and cART initiation than those without VF [median 49 (IQR 27–64) versus 15 (IQR 1–38) months, respectively; P = 0.015]. The median total peripheral blood mononuclear cell (PBMC) HIV DNA copy number was higher in patients with VF than in those without VF [417 (range 85–4151) versus 147 (range 16–4132) copies/106 PBMCs, respectively; P = 0.022]. Conclusions: A lower CD4 nadir, a longer time between HIV diagnosis and cART initiation, and a higher HIV DNA copy number at the time of DTG monotherapy initiation were associated with VF. While there clearly is no future role for DTG monotherapy, ongoing and future studies on the efficacy of maintenance dual therapy (e.g. DTG lamivudine) may have to take these variables into account in their study design and analysis