RESUMO
The HIV genotypic resistance test (GRT) is a standard of care for the clinical management of HIV/AIDS patients. In recent decades, population or Sanger sequencing has been the foundation for drug resistance monitoring in clinical settings. However, the advent of high-throughput or next-generation sequencing has caused a paradigm shift towards the detection and characterization of low-abundance covert mutations that would otherwise be missed by population sequencing. This is clinically significant, as these mutations can potentially compromise the efficacy of antiretroviral therapy, causing poor virologic suppression. Therefore, it is important to develop a more sensitive method so as to reliably detect clinically actionable drug-resistant mutations (DRMs). Here, we evaluated the diagnostic performance of a laboratory-developed, high-throughput, sequencing-based GRT using 103 archived clinical samples that were previously tested for drug resistance using population sequencing. As expected, high-throughput sequencing found all the DRMs that were detectable by population sequencing. Significantly, 78 additional DRMs were identified only by high-throughput sequencing, which is statistically significant based on McNemar's test. Overall, our results complement previous studies, supporting the notion that the two methods are well correlated, and the high-throughput sequencing method appears to be an excellent alternative for drug resistance testing in a clinical setting.
Assuntos
Fármacos Anti-HIV , Infecções por HIV , Soropositividade para HIV , HIV-1 , Humanos , HIV-1/genética , Farmacorresistência Viral/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Genótipo , Mutação , Fármacos Anti-HIV/farmacologia , Fármacos Anti-HIV/uso terapêuticoRESUMO
BACKGROUND: The British Antarctic bases offer a semiclosed environment for assessing the transmission and persistence of seasonal respiratory viruses. METHODS: Weekly swabbing was performed for respiratory pathogen surveillance (including SARS-CoV-2), at 2 British Antarctic Survey bases, during 2020: King Edward Point (KEP, 30 June to 29 September, 9 participants, 124 swabs) and Rothera (9 May to 6 June, 27 participants, 127 swabs). Symptom questionnaires were collected for any newly symptomatic cases that presented during this weekly swabbing period. RESULTS: At KEP, swabs tested positive for non-SARS-CoV-2 seasonal coronavirus (2), adenovirus (1), parainfluenza 3 (1), and respiratory syncytial virus B (1). At Rothera, swabs tested positive for non-SARS-CoV-2 seasonal coronavirus (3), adenovirus (2), parainfluenza 4 (1), and human metapneumovirus (1). All bacterial agents identified were considered to be colonizers and not pathogenic. CONCLUSIONS: At KEP, the timeline indicated that the parainfluenza 3 and adenovirus infections could have been linked to some of the symptomatic cases that presented. For the other viruses, the only other possible sources were the visiting ship crew members. At Rothera, the single symptomatic case presented too early for this to be linked to the subsequent viral detections, and the only other possible source could have been a single nonparticipating staff member.
Assuntos
Infecções por Adenoviridae , COVID-19 , Infecções por Paramyxoviridae , Infecções Respiratórias , Vírus , Humanos , COVID-19/epidemiologia , Pandemias , SARS-CoV-2 , Estudos Prospectivos , Regiões Antárticas , Infecções por Paramyxoviridae/epidemiologia , Inquéritos e QuestionáriosRESUMO
During the "first wave" of the coronavirus disease 2019 (COVID-19) pandemic in the United Kingdom (March-June 2020), the city of Leicester was particularly hard hit, resulting in reimposed lockdown measures. Although initial polymerase chain reaction testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was attempted within the community, testing was soon abandoned due to an inability to keep up with demand by local laboratories. It is therefore feasible that undiagnosed transmission of COVID-19 in the community by asymptomatic individuals was a real possibility. Therefore, retrospective SARS-CoV-2 immunoglobulin G (IgG) testing of archived sera from out-patients visiting University Hospitals of Leicester NHS Trust service was performed to investigate the transmission of SARS-CoV-2 in the community. A total of 1779 sera samples were tested from samples collected between 16th March and 3rd June 2020, of which 202 (11.35%) were SARS-CoV-2 IgG positive. Positivity was lowest in March (2.54%) at the beginning of the pandemic before peaking in April (17.16%) before a decline in May and June (11.16% and 12.68%, respectively). This retrospective screening offers some insight into the early patterns of SARS-CoV-2 transmission within a sampled community population during the first wave of the COVID-19 pandemic; supporting the argument for more community screening during high incidences of pandemics.
Assuntos
Anticorpos Antivirais/sangue , COVID-19/diagnóstico , COVID-19/epidemiologia , Imunoglobulina G/sangue , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Feminino , Humanos , Testes Imunológicos , Lactente , Recém-Nascido , Masculino , Programas de Rastreamento/estatística & dados numéricos , Pessoa de Meia-Idade , Pandemias , Estudos Retrospectivos , Reino Unido/epidemiologia , Adulto JovemRESUMO
To access temporal changes in psychobehavioral responses to the coronavirus disease (COVID-19) pandemic, we conducted a 5-round (R1-R5) longitudinal population-based online survey in Hong Kong during January-September 2020. Most respondents reported wearing masks (R1 99.0% to R5 99.8%) and performing hand hygiene (R1 95.8% to R5 97.7%). Perceived COVID-19 severity decreased significantly, from 97.4% (R1) to 77.2% (R5), but perceived self-susceptibility remained high (87.2%-92.8%). Female sex and anxiety were associated with greater adoption of social distancing. Intention to receive COVID-19 vaccines decreased significantly (R4 48.7% to R5 37.6%). Greater anxiety, confidence in vaccine, and collective responsibility and weaker complacency were associated with higher tendency to receive COVID-19 vaccines. Although its generalizability should be assumed with caution, this study helps to formulate health communication strategies and foretells the initial low uptake rate of COVID-19 vaccines, suggesting that social distancing should be maintained in the medium term.
Assuntos
COVID-19 , Vacinas , Vacinas contra COVID-19 , Feminino , Hong Kong/epidemiologia , Humanos , Pandemias/prevenção & controle , SARS-CoV-2RESUMO
As the COVID-19 pandemic continues, the availability of several different new vaccines, their varying supply levels, effectiveness, and immunity duration across different ethnic populations, together with natural infection rates, will have an impact on when each country can reach herd immunity (ranging from 15.3% to 77.1%). Here we estimate the population proportions still required to gain immunity (ranging from 0.01% to 48.8%) to reach an overall herd immunity level to stop the exponential virus spread in 32 selected countries.
Assuntos
Vacinas contra COVID-19 , COVID-19 , Imunidade Coletiva , COVID-19/imunologia , COVID-19/prevenção & controle , Humanos , Pandemias/prevenção & controleRESUMO
We performed a retrospective screening of 428 serum samples for anti-SARS-CoV-2 immunoglobulin during a period of low prevalence. Employing two different serological tests yielded discrepant results for 10 samples; highlighting an increased risk of potential false positive results and the need for further confirmatory testing before publication of data.
Assuntos
COVID-19 , SARS-CoV-2 , Brasil , Reações Falso-Positivas , Humanos , Imunoglobulinas , Prevalência , Estudos Retrospectivos , Sensibilidade e EspecificidadeRESUMO
Coronavirus disease 2019 (COVID-19) is generally a relatively mild illness in children. An emerging disease entity coined as pediatric inflammatory multisystem syndrome temporally associated with SARS-CoV-2 (PIMS-TS) has been reported recently, but is very rare and only affects a very small minority of children. Here we describe the clinical presentations and outcomes of three teenagers with serologically-confirmed SARS-CoV-2 infection admitted to a pediatric intensive care unit for PIMS-TS. Although their initial presentations were very similar, their COVID-19-related disease varied in severity.
Assuntos
COVID-19/diagnóstico , COVID-19/fisiopatologia , Síndrome de Resposta Inflamatória Sistêmica/diagnóstico , Síndrome de Resposta Inflamatória Sistêmica/fisiopatologia , Adolescente , COVID-19/terapia , Teste Sorológico para COVID-19 , Feminino , Humanos , Unidades de Terapia Intensiva/estatística & dados numéricos , Masculino , Índice de Gravidade de Doença , Síndrome de Resposta Inflamatória Sistêmica/terapia , Reino UnidoRESUMO
BackgroundCOVID-19, caused by SARS-CoV-2, first appeared in China and subsequently developed into an ongoing epidemic. Understanding epidemiological factors characterising the transmission dynamics of this disease is of fundamental importance.AimsThis study aimed to describe key epidemiological parameters of COVID-19 in Hong Kong.MethodsWe extracted data of confirmed COVID-19 cases and their close contacts from the publicly available information released by the Hong Kong Centre for Health Protection. We used doubly interval censored likelihood to estimate containment delay and serial interval, by fitting gamma, lognormal and Weibull distributions to respective empirical values using Bayesian framework with right truncation. A generalised linear regression model was employed to identify factors associated with containment delay. Secondary attack rate was also estimated.ResultsThe empirical containment delay was 6.39 days; whereas after adjusting for right truncation with the best-fit Weibull distribution, it was 10.4 days (95% CrI: 7.15 to 19.81). Containment delay increased significantly over time. Local source of infection and number of doctor consultations before isolation were associated with longer containment delay. The empirical serial interval was 4.58-6.06 days; whereas the best-fit lognormal distribution to 26 certain-and-probable infector-infectee paired data gave an estimate of 4.77 days (95% CrI: 3.47 to 6.90) with right-truncation. The secondary attack rate among close contacts was 11.7%.ConclusionWith a considerable containment delay and short serial interval, contact-tracing effectiveness may not be optimised to halt the transmission with rapid generations replacement. Our study highlights the transmission risk of social interaction and pivotal role of physical distancing in suppressing the epidemic.
Assuntos
Busca de Comunicante , Infecções por Coronavirus , Monitoramento Epidemiológico , Pandemias , Pneumonia Viral , Adulto , Idoso , Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , COVID-19 , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/transmissão , Feminino , Hong Kong/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Isolamento de Pacientes , Pneumonia Viral/diagnóstico , Pneumonia Viral/epidemiologia , Pneumonia Viral/transmissão , Encaminhamento e Consulta , SARS-CoV-2 , Fatores de Tempo , Adulto JovemRESUMO
Close contact was first identified as the primary route of transmission for most respiratory infections in the early 20th century. In this review, we synthesize the existing understanding of the mechanisms of close contact transmission. We focus on two issues: the mechanism of transmission in close contact, namely the transmission of the expired particles between two people, and the physical parameters of close contact that affect the exposure of particles from one individual to another, or how the nature of close contact plays a role in transmission. We propose the existence of three sub-routes of transmission: short-range airborne, large droplets, and immediate body-surface contact. We also distinguish a "body contact," which is defined with an interpersonal distance of zero, from a close contact. We demonstrate herein that the short-range airborne sub-route may be most common. The timescales over which data should be collected to assess the transmission risk during close contact events are much shorter than those required for the distant airborne or fomite routes. The current paucity of high-resolution data over short distances and timescales makes it very difficult to assess the risk of infection in these circumstances.
Assuntos
Poluição do Ar em Ambientes Fechados/estatística & dados numéricos , Infecções Respiratórias/transmissão , Expiração , HumanosAssuntos
Infecções por Coronavirus , Coronavirus , Pneumonia Viral , Betacoronavirus , COVID-19 , China , Hong Kong , Humanos , Pandemias , SARS-CoV-2RESUMO
BACKGROUND: Human parechovirus (HPeV), like enteroviruses, usually causes mild self-limiting respiratory and gastrointestinal symptoms. In infants, HPeV can occasionally cause serious illnesses, including sepsis-like syndrome and encephalitis. In summer 2016, Public Health England (PHE) received increasing reports of severe HPeV infections nationally. We, therefore, reviewed all infants with confirmed HPeV across England during 2016. METHODS: HPeV cases in infants aged <12 months reported to PHE during 2016 were followed up using a clinical questionnaire. Additional cases identified by clinicians completing the questionnaire were also included. RESULTS: We identified 106 infants with confirmed HPeV infection during 2016. The disease peaked during early summer. Most infants (98/106, 92%) were aged <90 days, and 43% (46/106) were neonates. Fever was the most commonly reported symptom (92%) and signs of circulatory shock were present in 53%. Eighteen infants (18%) required paediatric intensive care admission. Most infants had normal or low C reactive protein concentrations (<10 mg/dL in 75%, <50 mg/dL in 98%). A lumbar puncture was performed in 98% of cases; 92% (33/36) of neonates and 93% (53/57) of older infants had normal white cell count in the cerebrospinal fluid (CSF). Nearly all reported cases (98%) were confirmed by CSF PCR. All infants survived, but five had ongoing seizures after hospital discharge. CONCLUSIONS: HPeV is an important cause of febrile illness in infants and can have severe clinical presentations. Early diagnosis may help reduce antimicrobial use, unnecessary investigations and prolonged hospitalisation. While prognosis remains favourable, some infants will develop long-term complications-paediatricians should ensure appropriate follow-up after discharge.
Assuntos
Febre/virologia , Parechovirus/isolamento & purificação , Infecções por Picornaviridae/diagnóstico , Infecções por Picornaviridae/epidemiologia , Sepse/virologia , Líquido Cefalorraquidiano/virologia , Diagnóstico Precoce , Inglaterra/epidemiologia , Feminino , Febre/epidemiologia , Humanos , Lactente , Recém-Nascido , Masculino , Parechovirus/patogenicidade , Infecções por Picornaviridae/tratamento farmacológico , Infecções por Picornaviridae/virologia , Vigilância da População , Estudos Prospectivos , Sepse/epidemiologiaRESUMO
Pyrexia of unknown origin (PUO) is defined as a temperature of >38.3°C that lasts for >3 weeks, where no cause can be found despite appropriate investigation. Existing protocols for the work-up of PUO can be extensive and costly, motivating the application of recent advances in molecular diagnostics to pathogen testing. There have been many reports describing various analytical methods and performance of metagenomic pathogen testing in clinical samples but the economics of it has been less well studied. This study pragmatically evaluates the feasibility of introducing metagenomic testing in this setting by assessing the relative cost of clinically-relevant strategies employing this investigative tool under various cost and performance scenarios using Singapore as a demonstration case, and assessing the price and performance benchmarks, which would need to be achieved for metagenomic testing to be potentially considered financially viable relative to the current diagnostic standard. This study has some important limitations: we examined only impact of introducing the metagenomic test to the overall diagnostic cost and excluded costs associated with hospitalization and makes assumptions about the performance of the routine diagnostic tests, limiting the cost of metagenomic test, and the lack of further work-up after positive pathogen detection by the metagenomic test. However, these assumptions were necessary to keep the model within reasonable limits. In spite of these, the simplified presentation lends itself to the illustration of the key insights of our paper. In general, we find the use of metagenomic testing as second-line investigation is effectively dominated, and that use of metagenomic testing at first-line would typically require higher rates of detection or lower cost than currently available in order to be justifiable purely as a cost-saving measure. We conclude that current conditions do not warrant a widespread rush to deploy metagenomic testing to resolve any and all uncertainty, but rather as a front-line technology that should be used in specific contexts, as a supplement to rather than a replacement for careful clinical judgement.
Assuntos
Análise Custo-Benefício , Febre/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala/economia , Bactérias/genética , Febre/economia , Febre/microbiologia , Humanos , MetagenômicaRESUMO
Owing to advancements in molecular diagnostics, recent years have seen an increasing number of laboratories adopting respiratory viral panels to detect respiratory pathogens. In December 2015, the NxTAG respiratory pathogen panel (NxTAG RPP) was approved by the United States Food and Drug Administration. We compared the clinical performance of this new assay with that of the xTAG respiratory viral panel (xTAG RVP) FAST v2 using 142 clinical samples and 12 external quality assessment samples. Discordant results were resolved by using a laboratory-developed respiratory viral panel. The NxTAG RPP achieved 100% concordant negative results and 86.6% concordant positive results. It detected one coronavirus 229E and eight influenza A/H3N2 viruses that were missed by the xTAG RVP FAST v2. On the other hand, the NxTAG RPP missed one enterovirus/rhinovirus and one metapneumovirus that were detected by FAST v2. Both panels correctly identified all the pathogens in the 12 external quality assessment samples. Overall, the NxTAG RPP demonstrated good diagnostic performance. Of note, it was better able to subtype the influenza A/H3N2 viruses compared with the xTAG RVP FAST v2.
Assuntos
Infecções Respiratórias/virologia , Viroses/virologia , Coronavirus/genética , Coronavirus/isolamento & purificação , Enterovirus/genética , Enterovirus/isolamento & purificação , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/isolamento & purificação , Metapneumovirus/genética , Metapneumovirus/isolamento & purificação , Reação em Cadeia da Polimerase Multiplex , RNA Viral/análise , Kit de Reagentes para Diagnóstico , Infecções Respiratórias/diagnóstico , Viroses/diagnósticoRESUMO
Accurate full-length genomic sequences are important for viral phylogenetic studies. We developed a targeted high-throughput whole genome sequencing (HT-WGS) method for influenza A viruses, which utilized an enzymatic cleavage-based approach, the Nextera XT DNA library preparation kit, for library preparation. The entire library preparation workflow was adapted for the Sentosa SX101, a liquid handling platform, to automate this labor-intensive step. As the enzymatic cleavage-based approach generates low coverage reads at both ends of the cleaved products, we corrected this loss of sequencing coverage at the termini by introducing modified primers during the targeted amplification step to generate full-length influenza A sequences with even coverage across the whole genome. Another challenge of targeted HTS is the risk of specimen-to-specimen cross-contamination during the library preparation step that results in the calling of false-positive minority variants. We included an in-run, negative system control to capture contamination reads that may be generated during the liquid handling procedures. The upper limits of 99.99% prediction intervals of the contamination rate were adopted as cut-off values of contamination reads. Here, 148 influenza A/H3N2 samples were sequenced using the HTS protocol and were compared against a Sanger-based sequencing method. Our data showed that the rate of specimen-to-specimen cross-contamination was highly significant in HTS.
Assuntos
Genoma Viral/genética , Vírus da Influenza A Subtipo H3N2/genética , Influenza Humana/genética , Sequenciamento Completo do Genoma/métodos , Sequência de Bases , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Vírus da Influenza A Subtipo H3N2/patogenicidade , Influenza Humana/virologia , Filogenia , Análise de Sequência de DNARESUMO
BACKGROUND: Pathogens are often not identified in severe community-acquired pneumonia (CAP), and the few studies using polymerase chain reaction (PCR) techniques for virus detection are from temperate countries. OBJECTIVE: This study assesses if PCR amplification improves virus and bacteria detection, and if viral infection contributes to mortality in severe CAP in a tropical setting, where respiratory pathogens have less well-defined seasonality. METHODS: In this cohort study of patients with severe CAP in an intensive care unit, endotracheal aspirates for intubated patients and nasopharyngeal swabs for non-intubated patients were sent for PCR amplification for respiratory viruses. Blood, endotracheal aspirates for intubated patients, and sputum for non-intubated patients were analysed using a multiplex PCR system for bacteria. RESULTS: Out of 100 patients, using predominantly cultures, bacteria were identified in 42 patients; PCR amplification increased this number to 55 patients. PCR amplification identified viruses in 32 patients. In total, only bacteria, only viruses, and both bacteria and viruses were found in 37, 14, and 18 patients, respectively. The commonest viruses were influenza A H1N1/2009 and rhinovirus; the commonest bacterium was Streptococcus pneumoniae. Hospital mortality rates for patients with no pathogens, bacterial infection, viral infection, and bacterial-viral co-infection were 16.1, 24.3, 0, and 5.6%, respectively (p = 0.10). On multivariable analysis, virus detection was associated with lower mortality (adjusted odds ratio 0.12, 95% confidence interval 0.2-0.99; p = 0.049). CONCLUSIONS: Viruses and bacteria were detected in 7 of 10 patients with severe CAP with the aid of PCR amplification. Viral infection appears to be independently associated with lower mortality.
Assuntos
Influenza Humana/diagnóstico , Reação em Cadeia da Polimerase Multiplex , Infecções por Picornaviridae/diagnóstico , Pneumonia Bacteriana/diagnóstico , Pneumonia Pneumocócica/diagnóstico , Pneumonia Viral/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Infecções Comunitárias Adquiridas/diagnóstico , Infecções Comunitárias Adquiridas/microbiologia , Infecções Comunitárias Adquiridas/mortalidade , Infecções Comunitárias Adquiridas/virologia , Feminino , Mortalidade Hospitalar , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Influenza Humana/mortalidade , Influenza Humana/virologia , Unidades de Terapia Intensiva , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Infecções por Picornaviridae/mortalidade , Infecções por Picornaviridae/virologia , Pneumonia Bacteriana/microbiologia , Pneumonia Bacteriana/mortalidade , Pneumonia Pneumocócica/microbiologia , Pneumonia Pneumocócica/mortalidade , Pneumonia Viral/mortalidade , Pneumonia Viral/virologia , Estudos Prospectivos , Rhinovirus/genética , Streptococcus pneumoniae/genéticaRESUMO
Epitopes are the main targets for specific antibodies in the host defense systems. Recent studies have shown that amino acid (aa) substitutions located within the influenza A/H3N2 hemagglutinin 1 (HA1) epitopes A-E, particularly in A and B, result in antigenic drift. Viruses with such drift mutations may have resulted in more severe influenza-related illness during influenza epidemics between late 2012 and early 2015. We sought to quantify vaccine mismatches in epitopes A-E of the HA1 protein, and correlate these with the severity of the patient's illness. The influenza A/H3N2 clinical samples were collected between April 2009 and November 2013 (n=206). Patients were clinically stratified into groups with mild, moderate, and severe influenza-like illness (ILI). The impact of the number of aa mismatches in each of epitopes A-E, gender, age groups (⩽18, 19-64, ⩾65 years), and comorbidities on the likelihood that patients would suffer moderate and/or severe ILI due to influenza A/H3N2 infection were assessed. A higher number of aa mismatches in epitope A between the vaccine and locally circulating viruses correlated with more severe influenza infection, although this correlation was most significant with pre-existing comorbidities. A practical application of this finding would be to monitor patients (especially those in high-risk groups) infected with such viruses more closely, as they are at increased risk of developing more serious disease. Epidemiologically, it was of interest to note that viruses from subclade 3A of Victoria/208 strain were not detected in Singapore between 2009 and 2012. By contrast, these viruses were detected at a prevalence of up to 40% in the 2011-2012 influenza seasons in other regions of the Northern and Southern hemispheres. Such findings support the rationale for more regionally customized seasonal influenza vaccine compositions to optimize the protection of the population against locally circulating virus strains.
Assuntos
Vírus da Influenza A Subtipo H3N2/genética , Influenza Humana/virologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Substituição de Aminoácidos , Criança , Pré-Escolar , Epitopos/genética , Feminino , Genes Virais , Humanos , Lactente , Vírus da Influenza A Subtipo H3N2/imunologia , Vírus da Influenza A Subtipo H3N2/patogenicidade , Influenza Humana/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Índice de Gravidade de Doença , Proteínas Virais/genética , Adulto JovemRESUMO
Adamantanes and neuraminidase inhibitors (NAIs) are two classes of antiviral drugs available for the chemoprophylaxis and treatment of influenza infections. To determine the frequency of drug resistance in influenza A/H3N2 viruses in Singapore, large-scale sequencing of neuraminidase (NA) and matrix protein (MP) genes was performed directly without initial culture amplification. 241 laboratory-confirmed influenza A/H3N2 clinical samples, collected between May 2009 and November 2013 were included. In total, 229 NA (95%) and 241 MP (100%) complete sequences were obtained. Drug resistance mutations in the NA and MP genes were interpreted according to published studies. For the NAIs, a visual inspection of the aligned NA sequences revealed no known drug resistant genotypes (DRGs). For the adamantanes, the well-recognised S31N DRG was identified in all 241 MP genes. In addition, there was an increasing number of viruses carrying the combination of D93G+Y155F+D251V (since May 2013) or D93G (since March 2011) mutations in the NA gene. However, in-vitro NAI testing indicated that neither D93G+Y155F+D251V nor D93G alone conferred any changes in NAI susceptibility. Lastly, an I222T mutation in the NA gene that has previously been reported to cause oseltamivir-resistance in influenza A/H1N1/2009, B, and A/H5N1, was detected from a treatment-naïve patient. Further in-vitro NAI testing is required to confirm the effect of this mutation in A/H3N2 virus.