RESUMO
BACKGROUND: Immunocompromised individuals can become chronically infected with norovirus, but effective antiviral therapies are not yet available. METHODS: Treatments with nitazoxanide, ribavirin, interferon alpha-2a, and nasoduodenally administered immunoglobulins were evaluated sequentially in an immunocompromised patient chronically infected with norovirus. In support, these components were also applied to measure norovirus inhibition in intestinal enteroid cultures in vitro. Viral RNA levels were determined in fecal and plasma samples during each treatment and viral genomes were sequenced. RESULTS: None of the antivirals resulted in a reduction of viral RNA levels in feces or plasma. However, during ribavirin treatment, there was an increased accumulation of virus genome mutations. In vitro, an effect of interferon alpha-2a on virus replication was observed and a genetically related strain was neutralized effectively in vitro using immunoglobulins and post-norovirus-infection antiserum. In agreement, after administration of immunoglobulins, the patient cleared the infection. CONCLUSIONS: Intestinal enteroid cultures provide a relevant system to evaluate antivirals and the neutralizing potential of immunoglobulins. We successfully treated a chronically infected patient with immunoglobulins, despite varying results reported by others. This case study provides in-depth, multifaceted exploration of norovirus treatment that can be used as a guidance for further research towards norovirus treatments.
Assuntos
Infecções por Caliciviridae , Imunodeficiência de Variável Comum , Norovirus , Humanos , Antivirais/uso terapêutico , Antivirais/farmacologia , Infecções por Caliciviridae/tratamento farmacológico , Imunodeficiência de Variável Comum/complicações , Imunodeficiência de Variável Comum/tratamento farmacológico , Imunoglobulinas , Interferon-alfa/uso terapêutico , Norovirus/genética , Ribavirina/uso terapêutico , Ribavirina/farmacologia , RNA Viral/genética , Replicação ViralRESUMO
During COVID-19 lockdown, the in-hospital number of HIV indicator conditions decreased disproportionally compared with other non-COVID-19 diseases, which was accompanied by reduced HIV testing rates, number and proportion of positive HIV tests, and new HIV referrals, with more late presentation after lockdown cessation, indicating a significantly impacted HIV care continuum.
Assuntos
COVID-19 , Infecções por HIV , Controle de Doenças Transmissíveis , Continuidade da Assistência ao Paciente , HIV , Infecções por HIV/tratamento farmacológico , Infecções por HIV/epidemiologia , Hospitais , Humanos , SARS-CoV-2RESUMO
Lower respiratory tract (LRT) disease induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can deteriorate to acute respiratory distress syndrome (ARDS). Because the release of neutrophil extracellular traps (NETs) is implicated in ARDS pathogenesis, we investigated the presence of NETs and correlates of pathogenesis in blood and LRT samples of critically ill patients with COVID-19. Plasma NET levels peaked early after intensive care unit admission and were correlated with the SARS-CoV-2 RNA load in sputum and levels of neutrophil-recruiting chemokines and inflammatory markers in plasma samples. The baseline plasma NET quantity was correlated with disease severity but was not associated with soluble markers of thrombosis or with development of thrombosis. High NET levels were present in LRT samples and persisted during the course of COVID-19, consistent with the detection of NETs in bronchi and alveolar spaces in lung tissue from deceased patient with COVID-19. Thus, NETs are produced and retained in the LRT of critically ill patients with COVID-19 and could contribute to SARS-CoV-2-induced ARDS disease.
Assuntos
Líquido da Lavagem Broncoalveolar/virologia , COVID-19/complicações , COVID-19/patologia , Armadilhas Extracelulares/virologia , Síndrome do Desconforto Respiratório/etiologia , Síndrome do Desconforto Respiratório/patologia , SARS-CoV-2 , Adulto , Idoso , Biomarcadores , Quimiocinas/sangue , Estudos de Coortes , Angiografia por Tomografia Computadorizada , Estado Terminal , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Países Baixos/epidemiologia , Estudos Prospectivos , Índice de Gravidade de Doença , Trombose/virologia , Carga ViralRESUMO
Background: A high genetic barrier to resistance to the integrase strand transfer inhibitor (INSTI) dolutegravir has been reported in vitro and in vivo. We describe the dynamics of INSTI resistance-associated mutations (INSTI-RAMs) and mutations in the 3'-polypurine tract (3'-PPT) in relation to virologic failure (VF) observed in the randomized Dolutegravir as Maintenance Monotherapy for HIV-1 study (DOMONO, NCT02401828). Methods: From 10 patients with VF, plasma samples were collected before the start of cART and during VF, and were used to generate Sanger sequences of integrase, the 5' terminal bases of the 3' long terminal repeat (LTR), and the 3'-PPT. Results: Median human immunodeficiency virus RNA load at VF was 3490 copies/mL (interquartile range 1440-4990 copies/mL). INSTI-RAMs (S230R, R263K, N155H, and E92Q+N155H) were detected in 4 patients, no INSTI-RAMs were detected in 4 patients, and sequencing of the integrase gene was unsuccessful in 2 patients. The time to VF ranged from 4 weeks to 72 weeks. In 1 patient, mutations developed in the highly conserved 3'-PPT. No changes in the terminal bases of the 3'-LTR were observed. Conclusions: The genetic barrier to resistance is too low to justify dolutegravir maintenance monotherapy because single INSTI-RAMs are sufficient to cause VF. The large variation in time to VF suggests that stochastic reactivation of a preexisting provirus containing a single INSTI-RAM is the mechanism for failure. Changes in the 3'-PPT point to a new dolutegravir resistance mechanism in vivo. Clinical Trials Registration: NCT02401828.
Assuntos
Farmacorresistência Viral , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Inibidores de Integrase de HIV/administração & dosagem , HIV-1/efeitos dos fármacos , Compostos Heterocíclicos com 3 Anéis/administração & dosagem , Mutação , Adulto , Feminino , HIV-1/isolamento & purificação , Humanos , Quimioterapia de Manutenção/métodos , Masculino , Pessoa de Meia-Idade , Oxazinas , Piperazinas , Piridonas , Ensaios Clínicos Controlados Aleatórios como Assunto , Análise de Sequência de DNA , Falha de Tratamento , Carga ViralRESUMO
Background: Dolutegravir (DTG) is an integrase strand-transfer inhibitor (INSTI) used for treatment of human immunodeficiency virus (HIV)-infected individuals. Owing to its high genetic barrier to resistance, DTG has been clinically investigated as maintenance monotherapy to maintain viral suppression and to reduce complication and healthcare costs. Our study aims to explain the underlying mechanism related to the emergence of a S230R substitution in patients who experienced virologic failure while using DTG monotherapy. Methods: We evaluated the effect of the S230R substitution in regard to integrase enzyme activity, viral infectivity, replicative capacity, and susceptibility to different INSTIs by biochemical and cell-based assays. Results: The S230R substitution conferred a 63% reduction in enzyme efficiency. S230R virus was 1.29-fold less infectious than wild-type virus but could replicate in PM1 cells without significant delay. Resistance levels against DTG, cabotegravir, raltegravir, and elvitegravir in tissue culture were 3.85-, 3.72-, 1.52-, and 1.21-fold, respectively, in virus with the S230R substitution. Conclusions: Our data indicate that the S230R substitution is comparable to the previously reported R263K substitution in some respects. Virologic failure during DTG monotherapy can occur through the development of the S230R or R263K mutation, without the need for high-level DTG resistance.
Assuntos
Substituição de Aminoácidos , Farmacorresistência Viral , Infecções por HIV/tratamento farmacológico , Inibidores de Integrase de HIV/uso terapêutico , HIV/efeitos dos fármacos , Compostos Heterocíclicos com 3 Anéis/uso terapêutico , Carga Viral , HIV/genética , HIV/crescimento & desenvolvimento , HIV/isolamento & purificação , Integrase de HIV/genética , Integrase de HIV/metabolismo , Humanos , Quimioterapia de Manutenção/métodos , Testes de Sensibilidade Microbiana , Mutação de Sentido Incorreto , Oxazinas , Piperazinas , Piridonas , Falha de Tratamento , Replicação ViralRESUMO
Classic human polyomaviruses (JC and BK viruses) become pathogenic when reactivating from latency. For the rare skin disease trichodysplasia spinulosa, we show that manifestations of the causative polyomavirus (TSPyV) occur during primary infection of the immunosuppressed host. High TSPyV loads in blood and cerebrospinal fluid, sometimes coinciding with cerebral lesions and neuroendocrine symptoms, marked the acute phase of trichodysplasia spinulosa, whereas initiation and maturation of TSPyV seroresponses occurred in the convalescent phase. TSPyV genomes lacked the rearrangements typical for reactivating polyomaviruses. These findings demonstrate the clinical importance of primary infection with this rapidly expanding group of human viruses and explain the rarity of some novel polyomavirus-associated diseases.
Assuntos
Hospedeiro Imunocomprometido , Infecções por Polyomavirus/patologia , Dermatopatias/virologia , Pele/patologia , Líquido Cefalorraquidiano/virologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Polyomavirus , Carga ViralRESUMO
Background: Treatment of blood samples from hemorrhagic fever virus (HFV)-infected patients with 0.1% detergents has been recommended for virus inactivation and subsequent safe laboratory testing. However, data on virus inactivation by this procedure are lacking. Here we show the effect of this procedure on diagnostic test results and infectious Ebola virus (EBOV) titers. Methods: Serum and whole-blood samples were treated with 0.1% or 1% sodium dodecyl sulfate (SDS) or 0.1% Triton X-100 and assayed for clinical chemistry and malaria antigen detection. Infectious EBOV titers were determined in SDS-treated plasma and whole blood from EBOV-infected nonhuman primates (NHPs). Infectious titers of EBOV or herpes simplex virus type 1 (HSV-1) in detergents-treated cell culture medium containing various serum concentrations were determined. Results: Laboratory test results were not affected by 0.1% detergent treatment of blood samples, in contrast with 1% SDS treatment. However, 0.1% detergent treatment did not inactivate EBOV in blood samples from infected NHPs. Experiments with cell culture medium showed that virus inactivation by detergents is annulled at physiological serum concentrations. Conclusions: Treatment of blood samples with 0.1% SDS or Triton X-100 does not inactivate EBOV. Inactivation protocols for HFV should be validated with serum and whole blood.
Assuntos
Detergentes/farmacologia , Ebolavirus/efeitos dos fármacos , Soro , Dodecilsulfato de Sódio/farmacologia , Inativação de Vírus/efeitos dos fármacos , Animais , Herpes Simples , Humanos , Laboratórios , Macaca mulatta , OctoxinolRESUMO
In October 2016, a severe infection with swine influenza A(H1N1) virus of the Eurasian avian lineage occurred in a child with a previous history of eczema in the Netherlands, following contact to pigs. The patient's condition deteriorated rapidly and required life support through extracorporeal membrane oxygenation. After start of oseltamivir treatment and removal of mucus plugs, the patient fully recovered. Monitoring of more than 80 close unprotected contacts revealed no secondary cases.
Assuntos
Oxigenação por Membrana Extracorpórea , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Influenza Humana/diagnóstico , Infecções Respiratórias/virologia , Síndrome Respiratória Aguda Grave/terapia , Animais , Antivirais/uso terapêutico , Humanos , Influenza Humana/tratamento farmacológico , Influenza Humana/virologia , Unidades de Terapia Intensiva Pediátrica , Países Baixos , Infecções por Orthomyxoviridae/transmissão , Infecções por Orthomyxoviridae/veterinária , Infecções por Orthomyxoviridae/virologia , Oseltamivir/uso terapêutico , Reação em Cadeia da Polimerase em Tempo Real , Infecções Respiratórias/diagnóstico , Infecções Respiratórias/tratamento farmacológico , Síndrome Respiratória Aguda Grave/complicações , Suínos , Doenças dos Suínos/transmissão , Doenças dos Suínos/virologia , Resultado do TratamentoAssuntos
Aborto Espontâneo/virologia , Líquido Amniótico/virologia , Embrião de Mamíferos/virologia , Complicações Infecciosas na Gravidez , Infecção por Zika virus/complicações , Zika virus/isolamento & purificação , Adulto , Feminino , Morte Fetal , Humanos , Placenta/patologia , Placenta/virologia , Gravidez , Primeiro Trimestre da Gravidez , ViremiaAssuntos
Soropositividade para HIV , HIV-1 , Candida , Compostos Heterocíclicos com 3 Anéis , Humanos , Oxazinas , Piperazinas , PiridonasRESUMO
Herpes simplex virus type 1 (HSV-1) is a neurotropic alphaherpesvirus that establishes a lifelong infection in sensory neurons of infected individuals, accompanied with intermittent reactivation of latent virus causing (a)symptomatic virus shedding. Whereas acyclovir (ACV) is a safe and highly effective antiviral to treat HSV-1 infections, long-term usage can lead to emergence of ACV resistant (ACVR) HSV-1 and subsequently ACV refractory disease. Here, we isolated an HSV-1 strain from a patient with reactivated herpetic eye disease that did not respond to ACV treatment. The isolate carried a novel non-synonymous F289S mutation in the viral UL23 gene encoding the thymidine kinase (TK) protein. Because ACV needs conversion by viral TK and subsequently cellular kinases to inhibit HSV-1 replication, the UL23 gene is commonly mutated in ACVR HSV-1 strains. The potential role of the F289S mutation causing ACVR was investigated using CRISPR/Cas9-mediated HSV-1 genome editing. Reverting the F289S mutation in the original clinical isolate to the wild-type sequence S289F resulted in an ACV-sensitive (ACVS) phenotype, and introduction of the F289S substitution in an ACVS HSV-1 reference strain led to an ACVR phenotype. In summary, we identified a new HSV-1 TK mutation in the eye of a patient with ACV refractory herpetic eye disease, which was identified as the causative ACVR mutation with the aid of CRISPR/Cas9-mediated genome engineering technology. Direct editing of clinical HSV-1 isolates by CRISPR/Cas9 is a powerful strategy to assess whether single residue substitutions are causative to a clinical ACVR phenotype.
Assuntos
Aciclovir , Antivirais , Sistemas CRISPR-Cas , Farmacorresistência Viral , Edição de Genes , Herpesvirus Humano 1 , Mutação , Timidina Quinase , Timidina Quinase/genética , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 1/enzimologia , Humanos , Farmacorresistência Viral/genética , Aciclovir/farmacologia , Aciclovir/uso terapêutico , Antivirais/farmacologia , Antivirais/uso terapêutico , Herpes Simples/virologia , Herpes Simples/tratamento farmacológicoRESUMO
BACKGROUND: The two main objectives were to evaluate the COVID-19 point prevalence and the test performance of the WHO case definition to diagnose COVID-19 clinically in people with HIV in West Ukraine. METHODS: Multicenter cross-sectional study in Lviv, Ukraine, from October 2020-November 2021. COVID-19 unvaccinated people with HIV were included regardless of COVID-19 symptoms at routine clinical visits and had standardized medical, quality of life (EQ(5D)) and SARS-CoV-2 serology assessments. Reported symptoms indicating potential COVID-19 events at inclusion or between March 2020 and inclusion were classified by the WHO case definition as suspected, probable or confirmed. A clinical COVID-19 case was defined as being SARS-CoV-2 seropositive with at least a suspected COVID-19 according to the WHO case definition. The primary endpoints were the clinical COVID-19 prevalence and the test characteristics of the WHO case definition with SARS-CoV-2 serology as reference. (Clinicaltrials.gov:NCT04711954). RESULTS: The 971 included people with HIV were median 40 years, 38.8% women, 44.8% had prior AIDS, and 55.6% had comorbidities. SARS-CoV-2 seroprevalence was 40.1% (95%CI:37.0-43.1) and 20.5% (95%CI:18.0-23.1) had clinical COVID-19 median 4 months (IQR:2-7) before inclusion. Clinical COVID-19 occurred less frequently in people with HIV with tuberculosis history, injecting drug use, CD4+ T-cells <200/mL and unemployment. The quality of life was not impacted after COVID-19. An at least probable COVID-19 classification by the WHO case definition had 44.1% sensitivity (95%CI:38.7-49.7), 85.2% specificity (95%CI:81.5-88.4), 66.6% positive predictive value (95%CI:59.8-73.0) and 69.5% negative predictive value (95%CI:65.5-73.3) to diagnose COVID-19. CONCLUSIONS: COVID-19 unvaccinated people with HIV from Ukraine had a significant COVID-19 rate and using the WHO case definition had insufficient diagnostic accuracy to diagnose these cases. The lower burden in vulnerable people with HIV was unexpected but might reflect a shielding effect.
Assuntos
Algoritmos , COVID-19 , Infecções por HIV , SARS-CoV-2 , Humanos , COVID-19/epidemiologia , COVID-19/diagnóstico , Ucrânia/epidemiologia , Feminino , Masculino , Adulto , Infecções por HIV/epidemiologia , Infecções por HIV/diagnóstico , Infecções por HIV/complicações , Estudos Transversais , Pessoa de Meia-Idade , Prevalência , Organização Mundial da Saúde , Qualidade de VidaRESUMO
Pneumocystis jirovecii pneumonia is the main cause of severe respiratory failure in patients with advanced HIV disease who do not receive P. jirovecii prophylaxis. Other aetiological agents may contribute to the respiratory failure in these patients, which is highlighted by the case described below: A patient with advanced HIV disease was treated for a dual-infection with pandemic influenza A (H1N1) and P. jirovecii. Initially, his condition improved, but deteriorated after the emergence of oseltamivir-resistant influenza virus. This is the first documented case of emergence of drug-resistant influenza virus in a patient infected with HIV with a pandemic influenza A (H1N1) and P. jirovecii double infection.
Assuntos
Antivirais/uso terapêutico , Infecções por HIV/tratamento farmacológico , HIV-1 , Vírus da Influenza A Subtipo H1N1 , Influenza Humana/tratamento farmacológico , Oseltamivir/uso terapêutico , Pneumonia por Pneumocystis/tratamento farmacológico , Adulto , Coinfecção , Farmacorresistência Viral , Infecções por HIV/virologia , Humanos , Influenza Humana/virologia , Masculino , Pneumocystis carinii , Pneumonia por Pneumocystis/virologiaRESUMO
Human metapneumovirus (HMPV) is one of the leading causes of respiratory illness (RI), primarily in infants. Worldwide, two genetic lineages (A and B) of HMPV are circulating that are antigenically distinct and can each be further divided into genetic sublineages. Surveillance combined with large-scale whole-genome sequencing studies of HMPV are scarce but would help to identify viral evolutionary dynamics. Here, we analyzed 130 whole HMPV genome sequences obtained from samples collected from individuals hospitalized with RI and partial fusion (n = 144) and attachment (n = 123) protein gene sequences obtained from samples collected from patients with RI visiting general practitioners between 2005 and 2021 in the Netherlands. Phylogenetic analyses demonstrated that HMPV continued to group in the four sublineages described in 2004 (A1, A2, B1, and B2). However, one sublineage (A1) was no longer detected in the Netherlands after 2006, while the others continued to evolve. No differences were observed in dominant (sub)lineages between samples obtained from patients with RI being hospitalized and those consulting general practitioners. In both populations, viruses of lineage A2 carrying a 180-nucleotide or 111-nucleotide duplication in the attachment protein gene became the most frequently detected genotypes. In the past, different names for the newly energing lineages have been proposed, demonstrating the need for a consistent naming convention. Here, criteria are proposed for the designation of new genetic lineages to aid in moving toward a systematic HMPV classification. IMPORTANCE Human metapneumovirus (HMPV) is one of the major causative agents of human respiratory tract infections. Monitoring of virus evolution could aid toward the development of new antiviral treatments or vaccine designs. Here, we studied HMPV evolution between 2005 and 2021, with viruses obtained from samples collected from hospitalized individuals and patients with respiratory infections consulting general practitioners. Phylogenetic analyses demonstrated that HMPV continued to group in the four previously described sublineages (A1, A2, B1, and B2). However, one sublineage (A1) was no longer detected after 2006, while the others continued to evolve. No differences were observed in dominant (sub)lineages between patients being hospitalized and those consulting general practitioners. In both populations, viruses of lineage A2 carrying a 180-nucleotide or 111-nucleotide duplication in the attachment protein gene became the most frequently detected genotypes. These data were used to propose criteria for the designation of new genetic lineages to aid toward a systematic HMPV classification.
Assuntos
Metapneumovirus , Infecções por Paramyxoviridae , Infecções Respiratórias , Lactente , Humanos , Metapneumovirus/genética , Infecções por Paramyxoviridae/epidemiologia , Filogenia , Variação Genética , Genótipo , NucleotídeosRESUMO
OBJECTIVE: Timely identification of acute or early HIV infection (AEHI) is important to help prevent onward transmission, and understanding the number of secondary infections resulting from individuals with AEHI is key to planning HIV prevention services and case finding. DESIGN: We performed a phylogenetic investigation of a dense sample of individuals with AEHI who took part in the Netherlands Cohort Study on Acute HIV infection (NOVA) in the Netherlands during 2015-2021. METHODS: Transmission clusters were identified using phylogenetic analyses based on HIV pol sequences. The Tamura-Nei model was used to estimate genetic distance. A number of 1000 bootstraps was used to check the reliability of clustering using maximum likelihood. A cluster was defined as having a bootstrap value of at least 95% and a genetic distance of at most 1.5%. Sensitivity analyses using different values for the bootstrap and genetic distance were performed to study the reproducibility of the clustering. RESULTS: Of the 156 participants included in NOVA between July 2015 and April 2021, 134 individuals for whom baseline characteristics and genotypic resistance data at baseline were available could be included. We identified 10 clusters, but the majority of persons (111/134) were not part of a cluster, suggesting mainly independent transmission events. CONCLUSION: Mainly independent transmission events among a study population consisting predominantly of MSM in a low-incidence high-resource setting is likely the result of active AEHI case finding and direct start of treatment, and the roll-out over recent years of preventive measures such as preexposure prophylaxis.
Assuntos
Infecções por HIV , Humanos , Masculino , Infecções por HIV/epidemiologia , Reprodutibilidade dos Testes , Estudos de Coortes , Filogenia , Surtos de Doenças/prevenção & controle , Homossexualidade Masculina , Análise por ConglomeradosRESUMO
Solid organ transplant recipients (SOTRs) are at high risk of human herpesvirus (HHV)-related morbidity and mortality due to the use of immunosuppressive therapy. We aim to increase awareness and understanding of HHV disease burden in SOTRs by providing an overview of current prevention and management strategies as described in the literature and guidelines. We discuss challenges in both prevention and treatment as well as future perspectives.
Assuntos
Herpes Simples , Infecções por Herpesviridae , Herpesvirus Humano 6 , Transplante de Órgãos , Humanos , Transplante de Órgãos/efeitos adversos , Infecções por Herpesviridae/tratamento farmacológico , Infecções por Herpesviridae/prevenção & controle , TransplantadosRESUMO
Reactivation of the latent HIV-1 reservoir is a first step toward triggering reservoir decay. Here, we investigated the impact of the BAF complex inhibitor pyrimethamine on the reservoir of people living with HIV-1 (PLWH). Twenty-eight PLWH on suppressive antiretroviral therapy were randomized (1:1:1:1 ratio) to receive pyrimethamine, valproic acid, both, or no intervention for 14 days. The primary end point was change in cell-associated unspliced (CA US) HIV-1 RNA at days 0 and 14. We observed a rapid, modest, and significant increase in (CA US) HIV-1 RNA in response to pyrimethamine exposure, which persisted throughout treatment and follow-up. Valproic acid treatment alone did not increase (CA US) HIV-1 RNA or augment the effect of pyrimethamine. Pyrimethamine treatment did not result in a reduction in the size of the inducible reservoir. These data demonstrate that the licensed drug pyrimethamine can be repurposed as a BAF complex inhibitor to reverse HIV-1 latency in vivo in PLWH, substantiating its potential advancement in clinical studies.
Assuntos
Infecções por HIV , HIV-1 , Humanos , Linfócitos T CD4-Positivos , Infecções por HIV/tratamento farmacológico , HIV-1/fisiologia , Pirimetamina/farmacologia , Pirimetamina/uso terapêutico , RNA , Ácido Valproico/farmacologia , Ativação Viral , Latência ViralRESUMO
Plasmid-encoded ß-lactamases are a major reason for antibiotic resistance in gram negative bacteria. These enzymes hydrolyze the ß-lactam ring structure of certain ß-lactam antibiotics, consequently leading to their inactivation. The clinical situation demands for specific first-line antibiotic therapy combined with a quick identification of bacterial strains and their antimicrobial susceptibility. Strategies for the identification of ß-lactamase activity are often cumbersome and usually lack sensitivity and specificity. The current work demonstrates that matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) is an ideal tool for these analytical investigations. Herein, we describe a fast and specific assay to determine ß-lactamase activity in bacterial lysates. The feasibility of the analytical read-out was demonstrated on a MALDI-triple quadrupole (QqQ) and a MALDI time-of-flight (TOF) instrument, and the results allow the comparison of both approaches. The assay specifically measures enzyme-mediated, time-dependent hydrolysis of the ß-lactam ring structure of penicillin G and ampicillin and inhibition of hydrolysis by clavulanic acid for clavulanic acid susceptible ß-lactamases. The assay is reproducible and builds the basis for future in-depth investigations of ß-lactamase activity in various bacterial strains by mass spectrometry.
Assuntos
Proteínas de Bactérias/química , Ensaios Enzimáticos/métodos , Proteínas de Escherichia coli/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , beta-Lactamases/química , Calibragem , Escherichia coli/enzimologia , Cinética , Penicilina G/análogos & derivados , Penicilina G/química , Padrões de Referência , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/normasRESUMO
Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is an emerging analytical tool for the analysis of molecules with molar masses below 1,000 Da; that is, small molecules. This technique offers rapid analysis, high sensitivity, low sample consumption, a relative high tolerance towards salts and buffers, and the possibility to store sample on the target plate. The successful application of the technique is, however, hampered by low molecular weight (LMW) matrix-derived interference signals and by poor reproducibility of signal intensities during quantitative analyses. In this review, we focus on the biomedical application of MALDI-MS for the analysis of small molecules and discuss its favorable properties and its challenges as well as strategies to improve the performance of the technique. Furthermore, practical aspects and applications are presented.
Assuntos
Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Humanos , Metabolômica/métodos , Peso Molecular , Preparações Farmacêuticas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/economia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/tendênciasRESUMO
Here we describe a SARS-CoV-2 variant with diminished amplification of the ORF ORF1ab target in the Cobas® dual-target SARS-CoV-2 assay resulting in a discrepancy of Ct-values (Ct-value 20.7 for the E-gene and Ct-value 30.2 for ORF1ab). Five unique nucleotide mutations were identified in ORF1ab: C11450A (nsp10) C14178T (RdRp), G15006T (RdRp), G18394T (Hel), and G20995T (Hel). This case highlights the importance of surveillance of genomic regions used in molecular diagnostics and the importance of the public release of target regions used to update commercial and in-house developed SARS-CoV-2 PCR tests. This work underpins the importance of using dual-targets in molecular diagnostic assays to limit the change of false-negative results due to primer and/or probe mismatches.