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BACKGROUND: Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) target genes by molecular methods has been chosen as the main approach to identify individuals with Coronavirus disease 2019 (COVID-19) infection. OBJECTIVES: In this study, we developed an open-source RNA standard-based real-time quantitative RT-PCR (RT-qPCR) assay for quantitative diagnostics of SARS-CoV-2 from nasopharynx, oropharynx, saliva and plasma samples. METHODS AND FINDINGS: We evaluated three SARS-CoV-2 target genes and selected the RNA-dependent RNA polymerase (RdRp) gene, given its better performance. To improve the efficiency of the assay, a primer gradient containing 25 primers forward and reverse concentration combinations was performed. The forward and reverse primer pairs with 400 nM and 500 nM concentrations, respectively, showed the highest sensitivity. The LOD95% was ~60 copies per reaction. From the four biological matrices tested, none of them interfered with the viral load measurement. Comparison with the AllplexTM 2019-nCoV assay (Seegene) demonstrated that our test presents 90% sensitivity and 100% specificity. MAIN CONCLUSIONS: We developed an efficient molecular method able to measure absolute SARS-CoV-2 viral load with high replicability, sensitivity and specificity in different clinical samples.
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COVID-19 , SARS-CoV-2 , Humanos , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Carga ViralRESUMO
BACKGROUND Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) target genes by molecular methods has been chosen as the main approach to identify individuals with Coronavirus disease 2019 (COVID-19) infection. OBJECTIVES In this study, we developed an open-source RNA standard-based real-time quantitative RT-PCR (RT-qPCR) assay for quantitative diagnostics of SARS-CoV-2 from nasopharynx, oropharynx, saliva and plasma samples. METHODS AND FINDINGS We evaluated three SARS-CoV-2 target genes and selected the RNA-dependent RNA polymerase (RdRp) gene, given its better performance. To improve the efficiency of the assay, a primer gradient containing 25 primers forward and reverse concentration combinations was performed. The forward and reverse primer pairs with 400 nM and 500 nM concentrations, respectively, showed the highest sensitivity. The LOD95% was ~60 copies per reaction. From the four biological matrices tested, none of them interfered with the viral load measurement. Comparison with the AllplexTM 2019-nCoV assay (Seegene) demonstrated that our test presents 90% sensitivity and 100% specificity. MAIN CONCLUSIONS We developed an efficient molecular method able to measure absolute SARS-CoV-2 viral load with high replicability, sensitivity and specificity in different clinical samples.
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Approximately 185 million people worldwide are chronically infected with hepatitis C virus (HCV). The first-wave of approved NS3 protease inhibitors (PIs) were Telaprevir and Boceprevir, which are currently discontinued. Simeprevir is a second-wave PI incorporated into the Brazilian hepatitis C treatment protocol. Drug resistance plays a key role in patients' treatment regimen. Here, we developed a simple phenotypic assay to evaluate the impact of resistance mutations in HCV NS3 protease to PIs, using a protein expression vector containing wild type NS3 protease domain and NS4A co-factor. We analyzed the impact of five resistance mutations (T54A, V36M, V158I, V170I and T54S+V170I) against Telaprevir, Boceprevir and Simeprevir. Protein purifications were performed with low cost methodology, and enzymatic inhibition assays were measured by FRET. We obtained recombinant proteases with detectable activity, and IC50 and fold change values for the evaluated PIs were determined. The variant T54A showed the highest reduction of susceptibility for the PIs, while the other four variants exhibited lower levels of reduced susceptibility. Interestingly, V170I showed 3.2-fold change for Simeprevir, a new evidence about this variant. These results emphasize the importance of enzymatic assays in phenotypic tests to determine which therapeutic regimen should be implemented.
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OBJECTIVES: Feline immunodeficiency virus (FIV) is a lentivirus that induces AIDS-like disease in cats. Some of the antiretroviral drugs available to treat patients with HIV type 1 are used to treat FIV-infected cats; however, antiretroviral therapy (ART) is not used in cats as a long-term treatment. In this study, the effects of long-term ART were evaluated in domestic cats treated initially with the nucleoside transcriptase reverse inhibitor (NTRI) zidovudine (AZT) over a period ranging from 5-6 years, followed by a regimen of the NTRI lamivudine (3TC) plus AZT over 3 years. METHODS: Viral load, sequencing of pol (reverse transcriptase [RT]) region and CD4:CD8 lymphocyte ratio were evaluated during and after treatment. Untreated cats were evaluated as a control group. RESULTS: CD4:CD8 ratios were lower, and uncharacterized resistance mutations were found in the RT region in the group of treated cats. A slight increase in viral load was observed in some cats after discontinuing treatment. CONCLUSIONS AND RELEVANCE: The data strongly suggest that treated cats were resistant to therapy, and uncharacterized resistance mutations in the RT gene of FIV were selected for by AZT. Few studies have been conducted to evaluate the effect of long-term antiretroviral therapy in cats. To date, resistance mutations have not been described in vivo.
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Linfócitos T CD8-Positivos , Doenças do Gato/tratamento farmacológico , Síndrome de Imunodeficiência Adquirida Felina/tratamento farmacológico , Vírus da Imunodeficiência Felina/efeitos dos fármacos , Lamivudina/uso terapêutico , Inibidores da Transcriptase Reversa/uso terapêutico , Animais , Gatos , Seguimentos , Dados de Sequência Molecular , Carga Viral , Zidovudina/uso terapêuticoRESUMO
BACKGROUND: An observational study was conducted in Maputo, Mozambique, to investigate trends in prevalence of HIV drug resistance (HIVDR) in antiretroviral (ART) naïve subjects initiating highly active antiretroviral treatment (HAART). METHODOLOGY/PRINCIPAL FINDINGS: To evaluate the pattern of drug resistance mutations (DRMs) found in adults on ART failing first-line HAART [patients with detectable viral load (VL)]. Untreated subjects [Group 1 (G1; n=99)] and 274 treated subjects with variable length of exposure to ARV´s [6-12 months, Group 2 (G2;n=93); 12-24 months, Group 3 (G3;n=81); >24 months (G4;n=100)] were enrolled. Virological and immunological failure (VF and IF) were measured based on viral load (VL) and T lymphocyte CD4+ cells (TCD4+) count and genotypic resistance was also performed. Major subtype found was C (untreated: n=66, 97,06%; treated: n=36, 91.7%). Maximum virological suppression was observed in G3, and significant differences intragroup were observed between VF and IF in G4 (p=0.022). Intergroup differences were observed between G3 and G4 for VF (p=0.023) and IF between G2 and G4 (p=0.0018). Viral suppression (<50 copies/ml) ranged from 84.9% to 90.1%, and concordant VL and DRM ranged from 25% to 57%. WHO cut-off for determining VF as given by 2010 guidelines (>5000 copies/ml) identified 50% of subjects carrying DRM compared to 100% when lower VL cut-off was used (<50 copies/ml). Length of exposure to ARVs was directly proportional to the complexity of DRM patterns. In Mozambique, VL suppression was achieved in 76% of individuals after 24 months on HAART. This is in agreement with WHO target for HIVDR prevention target (70%). CONCLUSIONS: We demonstrated that the best way to determine therapeutic failure is VL compared to CD4 counts. The rationalized use of VL testing is needed to ensure timely detection of treatment failures preventing the occurrence of TDR and new infections.
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Antirretrovirais/uso terapêutico , Terapia Antirretroviral de Alta Atividade , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Adulto , Instituições de Assistência Ambulatorial , Contagem de Linfócito CD4 , Farmacorresistência Viral/genética , Feminino , Genótipo , Infecções por HIV/epidemiologia , Infecções por HIV/virologia , HIV-1/genética , HIV-1/fisiologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Masculino , Moçambique/epidemiologia , Mutação , Prevalência , Fatores de Tempo , Falha de Tratamento , Carga Viral/efeitos dos fármacos , Carga Viral/genéticaRESUMO
Background: An observational study was conducted in Maputo, Mozambique, to investigate trends in prevalence of HIV drug resistance (HIVDR) in antiretroviral (ART) naïve subjects initiating highly active antiretroviral treatment (HAART). Methodology/principal findings: To evaluate the pattern of drug resistance mutations (DRMs) found in adults on ART failing first-line HAART [patients with detectable viral load (VL)]. Untreated subjects [Group 1 (G1; n=99)] and 274 treated subjects with variable length of exposure to ARV´s [6-12 months, Group 2 (G2;n=93); 12-24 months, Group 3 (G3;n=81); >24 months (G4;n=100)] were enrolled. Virological and immunological failure (VF and IF) were measured based on viral load (VL) and T lymphocyte CD4+ cells (TCD4+) count and genotypic resistance was also performed. Major subtype found was C (untreated: n=66, 97,06%; treated: n=36, 91.7%). Maximum virological suppression was observed in G3, and significant differences intragroup were observed between VF and IF in G4 (p=0.022). Intergroup differences were observed between G3 and G4 for VF (p=0.023) and IF between G2 and G4 (p=0.0018). Viral suppression (<50 copies/ml) ranged from 84.9% to 90.1%, and concordant VL and DRM ranged from 25% to 57%. WHO cut-off for determining VF as given by 2010 guidelines (>5000 copies/ml) identified 50% of subjects carrying DRM compared to 100% when lower VL cut-off was used (<50 copies/ml). Length of exposure to ARVs was directly proportional to the complexity of DRM patterns. In Mozambique, VL suppression was achieved in 76% of individuals after 24 months on HAART. This is in agreement with WHO target for HIVDR prevention target (70%). Conclusions: We demonstrated that the best way to determine therapeutic failure is VL compared to CD4 counts. The rationalized use of VL testing is needed to ensure timely detection of treatment failures preventing the occurrence of TDR and new infections.
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Humanos , Animais , Masculino , Feminino , Adulto , Infecções por HIV/reabilitação , HIV-1/efeitos dos fármacos , Terapia Antirretroviral de Alta Atividade , Antirretrovirais/uso terapêutico , Infecções por HIV/epidemiologia , Infecções por HIV/virologia , Prevalência , HIV-1/fisiologia , HIV-1/genética , Falha de Tratamento , Contagem de Linfócito CD4 , Carga Viral/efeitos da radiação , Carga Viral/genética , Farmacorresistência Viral , Farmacorresistência Viral/genética , Interações Hospedeiro-Patógeno , Assistência Ambulatorial , Genótipo , MutaçãoRESUMO
Ipecac alkaloids are secondary metabolites produced in the medicinal plant Psychotria ipecacuanha. Emetine is the main alkaloid of ipecac and one of the active compounds in syrup of Ipecac with emetic property. Here we evaluated emetine's potential as an antiviral agent against Human Immunodeficiency Virus. We performed in vitro Reverse Transcriptase (RT) Assay and Natural Endogenous Reverse Transcriptase Activity Assay (NERT) to evaluate HIV RT inhibition. Emetine molecular docking on HIV-1 RT was also analyzed. Phenotypic assays were performed in non-lymphocytic and in Peripheral Blood Mononuclear Cells (PBMC) with HIV-1 wild-type and HIV-harboring RT-resistant mutation to Nucleoside Reverse Transcriptase Inhibitors (M184V). Our results showed that HIV-1 RT was blocked in the presence of emetine in both models: in vitro reactions with isolated HIV-1 RT and intravirion, measured by NERT. Emetine revealed a strong potential of inhibiting HIV-1 replication in both cellular models, reaching 80% of reduction in HIV-1 infection, with low cytotoxic effect. Emetine also blocked HIV-1 infection of RT M184V mutant. These results suggest that emetine is able to penetrate in intact HIV particles, and bind and block reverse transcription reaction, suggesting that it can be used as anti-HIV microbicide. Taken together, our findings provide additional pharmacological information on the potential therapeutic effects of emetine.
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Alcaloides/administração & dosagem , Emetina/administração & dosagem , Infecções por HIV/tratamento farmacológico , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Alcaloides/química , Fármacos Anti-HIV/administração & dosagem , Fármacos Anti-HIV/química , Emetina/química , Infecções por HIV/enzimologia , Infecções por HIV/virologia , Transcriptase Reversa do HIV/química , HIV-1/patogenicidade , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/virologia , Mutação , Inibidores da Transcriptase Reversa/administração & dosagem , Inibidores da Transcriptase Reversa/química , Replicação Viral/efeitos dos fármacosRESUMO
The ability of HIV to establish long-lived latent infection is mainly due to transcriptional silencing of viral genome in resting memory T lymphocytes. Here, we show that new semi-synthetic ingenol esters reactivate latent HIV reservoirs. Amongst the tested compounds, 3-caproyl-ingenol (ING B) was more potent in reactivating latent HIV than known activators such as SAHA, ingenol 3,20-dibenzoate, TNF-α, PMA and HMBA. ING B activated PKC isoforms followed by NF-κB nuclear translocation. As virus reactivation is dependent on intact NF-κB binding sites in the LTR promoter region ING B, we have shown that. ING B was able to reactivate virus transcription in primary HIV-infected resting cells up to 12 fold and up to 25 fold in combination with SAHA. Additionally, ING B promoted up-regulation of P-TEFb subunits CDK9/Cyclin T1. The role of ING B on promoting both transcription initiation and elongation makes this compound a strong candidate for an anti-HIV latency drug combined with suppressive HAART.
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Diterpenos/metabolismo , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Ativação Viral/efeitos dos fármacos , Latência Viral/efeitos dos fármacos , Linhagem Celular , Ésteres/metabolismo , Humanos , Replicação Viral/efeitos dos fármacosRESUMO
OBJECTIVE: In Mozambique, highly active antiretroviral treatment (HAART) was introduced in 2004 followed by decentralization and expansion, resulting in a more than 20-fold increase in coverage by 2009. Implementation of HIV drug resistance threshold surveys (HIVDR-TS) is crucial in order to monitor the emergence of transmitted viral resistance, and to produce evidence-based recommendations to support antiretroviral (ARV) policy in Mozambique. METHODS: World Health Organization (WHO) methodology was used to evaluate transmitted drug resistance (TDR) in newly diagnosed HIV-1 infected pregnant women attending ante-natal clinics in Maputo and Beira to non-nucleoside reverse transcriptase inhibitors (NNRTI), nucleoside reverse transcriptase inhibitors (NRTI) and protease inhibitors (PI). Subtypes were assigned using REGA HIV-1 subtyping tool and phylogenetic trees constructed using MEGA version 5. RESULTS: Although mutations associated with resistance to all three drug were detected in these surveys, transmitted resistance was analyzed and classified as <5% in Maputo in both surveys for all three drug classes. Transmitted resistance to NNRTI in Beira in 2009 was classified between 5-15%, an increase from 2007 when no NNRTI mutations were found. All sequences clustered with subtype C. CONCLUSIONS: Our results show that the epidemic is dominated by subtype C, where the first-line option based on two NRTI and one NNRTI is still effective for treatment of HIV infection, but intermediate levels of TDR found in Beira reinforce the need for constant evaluation with continuing treatment expansion in Mozambique.
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Fármacos Anti-HIV/farmacologia , Farmacorresistência Viral , Genótipo , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/genética , Adolescente , Adulto , Fármacos Anti-HIV/uso terapêutico , Terapia Antirretroviral de Alta Atividade , Farmacorresistência Viral/genética , Feminino , Infecções por HIV/tratamento farmacológico , Infecções por HIV/epidemiologia , HIV-1/classificação , Humanos , Masculino , Moçambique/epidemiologia , Mutação , Filogenia , Gravidez , Adulto JovemRESUMO
OBJECTIVE: To evaluate the polymorphisms and resistance mutations in gp41 HR1 region of HIV-1. METHODS: The study included 28 HIV-positive patients undergoing enfuvirtide (ENF) treatment or not from Porto Alegre, Rio Grande do Sul state, and Rio de Janeiro, Rio de Janeiro state, between 2006 and 2009. Resistance mutations and polymorphisms of the gp41 HR1 region were detected using the genomic DNA of 12 ENF-untreated patients and 16 patients in ENF treatment, encompassing subtypes B, C, and F1. Sample subtypes were determined by neighbor-joining phylogenetic analysis with a Kimura's two-parameter correction. RESULTS: A high prevalence of polymorphisms unrelated to resistance was observed. Among ENF-untreated patients, 16% showed mutations related with resistance. Among patients in ENF treatment, 50% had resistance-related mutations. Overall, 17% of all isolates showed the N42S polymorphism related to ENF hypersusceptibility. The presence of ENF resistance mutations in the group of treated patients reduced viral load. The V38A substitution was the most frequent among treatment-experienced patients followed by the G36D/E, N42D, and V38M substitutions. CONCLUSIONS: The V38A substitution in the gp41 HR region was the most common resistance mutation among ENF-treated patients and was associated with increased viral load.
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Fármacos Anti-HIV/farmacologia , Farmacorresistência Viral , Proteína gp41 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/farmacologia , Infecções por HIV/virologia , HIV-1/genética , Fragmentos de Peptídeos/farmacologia , Polimorfismo Genético , Substituição de Aminoácidos/genética , Fármacos Anti-HIV/uso terapêutico , Análise por Conglomerados , Enfuvirtida , Genótipo , Proteína gp41 do Envelope de HIV/uso terapêutico , Infecções por HIV/tratamento farmacológico , HIV-1/isolamento & purificação , Humanos , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Fragmentos de Peptídeos/uso terapêutico , Filogenia , Análise de Sequência de DNARESUMO
This work evaluated HIV-1 subtypes from different geographic regions and phenotypic data from drug-naïve HIV-positive pregnant women from Mozambique. We analyzed 75 pol sequences from patients and the distribution of the subtypes in three regions of Mozambique and found that the majority of samples analyzed clustered with subtype C. In the northern region, multiple variants were found 5 (approximately 18%) subtype A, 3 (approximately 11%) subtype D and 2 (approximately 7.1%) mosaics (A/C/D and C/D), whereas 18 (64.3%) isolates were subtype C, from a total of 28 samples. Already in the southern region, only one (5%) isolate of 20 samples was subtype D, and the other 19 (95%) isolates were subtype C. All 27 (100%) isolates from the central region grouped within clade C. No primary resistance mutations to IP, NNRTI or NRTI were found. There was no evidence of phenotypic resistance in any of the isolates tested, suggesting that neither the polymorphism in the protease, nor the one found at codon 215 of the RT gene caused an increase in phenotypic resistance. This finding suggests that HAART regimens indicated by WHO will probably be successful in Mozambique.
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Infecções por HIV/virologia , HIV-1/genética , HIV-1/isolamento & purificação , Farmacorresistência Viral , Feminino , Genótipo , Infecções por HIV/epidemiologia , HIV-1/classificação , Humanos , Moçambique/epidemiologia , Fenótipo , Gravidez , Produtos do Gene pol do Vírus da Imunodeficiência Humana/genéticaRESUMO
Candida yeasts frequently cause life-threatening systemic infections in immunocompromised hosts. In the present study, gelatin-SDS-PAGE analysis was used to characterize extracellular proteinases in 44 oral clinical isolates of Candida albicans from HIV-positive (29/50) and healthy children (15/50). Our survey indicates that these oral clinical isolates of C. albicans have complex extracellular proteolytic activity profiles, which illustrates the heterogeneity of this species. We showed four distinct proteolytic patterns composed of distinct serine (30-58 kDa) and metalloproteinase (64-95 kDa) activities, based on the inhibition profile with phenylmethylsulfonyl fluoride and 1,10-phenanthroline, respectively. This is the first report on secreted serine and metalloproteinases present in the culture supernatant fluids of C. albicans; however, we did not observe a significant correlation between proteolytic profile expressed by the C. albicans isolates from HIV-positive children and CD4(+) T cell count and plasma viral load.
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Infecções Oportunistas Relacionadas com a AIDS/microbiologia , Candida albicans/enzimologia , Candidíase Bucal/microbiologia , Metaloproteases/metabolismo , Serina Endopeptidases/metabolismo , Contagem de Linfócito CD4 , Candida albicans/classificação , Candida albicans/crescimento & desenvolvimento , Candida albicans/isolamento & purificação , Criança , Pré-Escolar , Meios de Cultivo Condicionados , Infecções por HIV/complicações , Infecções por HIV/virologia , HIV-1/fisiologia , Humanos , Lactente , Metaloproteases/classificação , Serina Endopeptidases/classificação , Carga ViralRESUMO
We have analyzed the effects of exogenous phospholipase C (PLC) on the cell-surface polypeptides and proteinases of Herpetomonas samuelpessoai grown in chemically defined conditions by SDS-PAGE gels. Live parasites treated with PLC released into the extracellular medium a complex profile of glycosylphosphatidylinositol (GPI)-anchored polypeptides ranging from 15 to 100 kDa, some of them with proteolytic activity. Two major metalloproteinases with apparent molecular masses of 63 and 115 kDa were observed after PLC hydrolysis. Interestingly, only the PLC-released soluble form of the 115-kDa metalloenzyme, and not the membrane-anchored form, displayed proteolytic activity, demonstrating that cleavage of the GPI anchor can lead to enzymatic activation.