Tze Ping Loh

Comparison of Mutation Patterns in Full-Genome A/H3N2 Influenza Sequences Obtained Directly from Clinical Samples and the Same Samples after a Single MDCK Passage

Human influenza viruses can be isolated efficiently from clinical samples using Madin-Darby canine kidney (MDCK) cells. However, this process is known to induce mutations in the virus as it adapts to this non-human cell-line. We performed a systematic study to record the pattern of MDCK-induced mutations observed across the whole influenza A/H3N2 genome. Seventy-seven clinical samples collected from 2009-2011 were included in the study. Two full influenza genomes were obtained for each sample: one from virus obtained directly from the clinical sample and one from the matching isolate cultured in MDCK cells. Comparison of the full-genome sequences obtained from each of these sources showed that 42% of the 77 isolates had acquired at least one MDCK-induced mutation. The presence or absence of these mutations was independent of viral load or sample origin (in-patients versus out-patients). Notably, all the five hemagglutinin missense mutations were observed at the hemaggutinin 1 domain only, particularly within or proximal to the receptor binding sites and antigenic site of the virus. Furthermore, 23% of the 77 isolates had undergone a MDCK-induced missense mutation, D151G/N, in the neuraminidase segment. This mutation has been found to be associated with reduced drug sensitivity towards the neuraminidase inhibitors and increased viral receptor binding efficiency to host cells. In contrast, none of the neuraminidase sequences obtained directly from the clinical samples contained the D151G/N mutation, suggesting that this mutation may be an indicator of MDCK culture-induced changes. These D151 mutations can confound the interpretation of the hemagglutination inhibition assay and neuraminidase inhibitor resistance results when these are based on MDCK isolates. Such isolates are currently in routine use in the WHO influenza vaccine and drug-resistance surveillance programs. Potential data interpretation miscalls can therefore be avoided by careful exclusion of such D151 mutants after further sequence analysis.

Impact of outpatient neuraminidase inhibitor treatment in patients infected with influenza A(H1N1)pdm09 at high risk of hospitalization: an Individual Participant Data (IPD) meta-analysis

Summary Our findings suggest that in populations at high risk for hospital admission, patients with laboratory-confirmed or clinically diagnosed A(H1N1)pdm09 infection, NAI treatment in the community or outpatient settings is associated with reduced likelihood of subsequent hospital admission.

Molecular Surveillance of Antiviral Drug Resistance of Influenza A/H3N2 Virus in Singapore, 2009-2013

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.

Clinical consequences of erroneous laboratory results that went unnoticed for 10 days

Erroneous laboratory results can adversely affect medical decisions. While the prevalence of laboratory errors has been well documented,1 their consequences, particularly when they go unnoticed, are less well reported. We describe the clinical consequences of a series of falsely elevated laboratory results that went unnoticed for 10 days. On 20 June 2012, an endocrinologist alerted the laboratory to possible spurious results after noticing extremely high insulin-like growth factor-1 (IGF-1) concentrations in two clinically asymptomatic patients. The ensuing investigations revealed an error in the analyser (Immulite 2000, Siemens, Surrey, UK) that performed five endocrine tests, including IGF-1, and affected all results reported on 11 June. The error eluded the internal quality control (QC) testing performed routinely prior to patient sample analysis. All erroneous results (n=63) were unknowingly reported. Retesting of all specimens belonging to 49 patients revealed 2.1- to >108-fold reductions in their results (see online supplementary table S1). The ranges of the erroneous results and the correct values (in parentheses), respectively, were: adrenocorticotrophic hormone (ACTH, n=10), 15.3–132 pmol/l ( 3000 IU/ml (n=15, 1000 IU/ml (n=7, <10–876); growth hormone, 2.12–188 µg/l (n=10, 0.35–9.63); IGF-1 603–2263 ng/ml (n=21, 54–292). All ordering physicians were immediately notified of the amended results. The engineers of the manufacturer were immediately brought in to conduct an extensive investigation, which included checking for reagent/probe/tubing contamination, reagent …

Glycated haemoglobin: what is the diagnostic yield at shortened testing intervals?

The proportion of sequential HbA1c exceeding critical difference (diagnostic yield), where it is considered clinically significant change, was calculated for different testing intervals. 12% and 26% of repeat HbA1c exceeded the critical difference before 30 and 90 days testing intervals, respectively. Repeating HbA1c within 4 weeks has poor diagnostic yield and should be avoided.

Predicting clinical severity based on substitutions near epitope A of influenza A/H3N2.

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 patients 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.

Emergence of G186D Mutation in the Presence of R292K Mutation in an Immunocompromised Child Infected with Influenza A/H3N2 Virus, Treated with Oseltamivir

ABSTRACT An immunocompromised child with influenza A/H3N2 virus infection, treated with oseltamivir from day 1, had nasal swabs taken on days 1, 4, 7, and 10 of the illness. Pyrosequencing showed increasing proportions of viruses with R292K (neuraminidase gene) and G186D (hemagglutinin gene) mutations, resulting in a viral load rebound by day 10.

Application of a High-Sensitivity Cardiac Troponin I Assay to a Health Screen Cohort of Young Asian Women and Association with Cardiovascular Risk Factors

To the Editor: The Global Task Force for the Universal Definition of Myocardial Infarction and the National Academy of Clinical Biochemistry recommend the 99th percentile of cardiac troponin as a diagnostic threshold, provided that assay precision can be demonstrated to have a CV of ≤10% at this concentration (1). To determine the 99th percentile, we applied a high-sensitivity cardiac troponin I (hscTnI) assay (Abbott Diagnostics) to an analysis of a cohort of young, apparently healthy Asian women with low cardiovascular risk, and correlated this result with demographic and clinical characteristics to identify factors associated with higher cTnI concentrations. The cohort was obtained from our hospital staff health-screening program. Fasting serum samples were stored at −70 °C for up to 6 months until analysis. Nonpregnant women 20–65 years of age with no self-reported personal history of heart disease, diabetes, or hypertension were selected for analysis of cTnI concentrations with the Abbott ARCHITECT i 4000 analyzer and a premarket prototype hscTnI assay. Institutional review board approval was obtained for this study (DSRB/2011/01931). SPSS version 19 (SPSS/IBM) was used for statistical analyses. We …

An in-house HIV genotyping assay for the detection of drug resistance mutations in Southeast Asian patients infected with HIV-1†

Genotyping for HIV drug resistance is costly and beyond the means for many Southeast Asian patients, who are self‐funded. This prompted the development of a more cost‐effective, in‐house assay for an ethnically diverse, Southeast Asian population at the National University Hospital in Singapore, using Sanger‐based sequencing. Plasma samples from 20 treatment‐failure patients with a broad spectrum of HIV drug resistance mutations were used to validate this assay clinically. Blinded testing gave concordant results for 7/7 (100%) protease drug resistance‐related mutations, including one major and six minor mutations, and 111/116 (95.7%) reverse‐transcriptase (RT) drug resistance‐related mutations, including 65 nucleoside RT inhibitors (NRTI) and 46 non‐nucleoside RT inhibitors (NNRTI) mutations. There were five discordant results, involving three NRTI‐ and two NNRTI‐resistance‐associated mutations. Highly conserved primers designed to have a wide coverage of the HIV pol gene (covering the entire protease and 395 codons of the RT region) enabled efficient multi‐ethnic population‐based genotyping. Reagents for this in‐house test cost around 60% less than those for commercially available assays (SGD150 vs. SGD350 per sample). In addition, this assay also identified mutations located within the C‐terminal domain (codons 312–560) of RT that are beyond the reach of most published and commercial GRTs. Currently, most research on C‐terminal drug‐resistance‐related mutations has been conducted on HIV subtype B infections. Therefore this assay enables further study of these C‐terminal mutations in Southeast Asian populations, where there is a high prevalence of CRF01_AE and other non‐subtype B HIV infections. J. Med. Virol. 84:394–401, 2012.

Meal rich in carbohydrate, but not protein or fat, reveals adverse immunometabolic responses associated with obesity.

BackgroundObesity-related insulin resistance is linked to inflammation. Immunometabolic function differs between lean and obese subjects, but whether macronutrient composition of ingested meals affects these responses is not well known. We examined the effects of a single meal rich in fat, protein, or carbohydrate on immunometabolic responses.MethodsNine lean insulin sensitive (LIS) men and 9 obese insulin resistant (OIR) men ingested high-carbohydrate (HC), high-fat (HF) or high-protein (HP) mixed meals in random order. We assessed plasma glucose, insulin, and cytokine responses and cytokine gene expression in circulating mononuclear cells (MNC) at fasting and postprandial states (up to 6-h).ResultsExpression of NF-κB and TNFα genes were greater; whereas that of TGFβ and IL-6 genes were lower, in the OIR compared to the LIS individuals. The differences were significantly greater after the HC meal, but not after the HP or HF meal. Similar results were obtained for plasma concentrations of TNFα and IL-6.ConclusionsOur findings indicate that a single HC meal has a distinct adverse effect on immunometabolic responses in the OIR individuals. The cumulative effect of such adverse responses to meals rich in carbohydrate may predispose the OIR individuals to a higher risk of cardiovascular disease.