Emergence of G186D mutation in the presence of R292K mutation in an immunocompromised child infected with influenza A/H3N2 virus, treated with oseltamivir.

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.

Adult invasive pneumococcal disease pre- and peri-pneumococcal conjugate vaccine introduction in a tertiary hospital in Singapore.

Streptococcus pneumoniae isolates causing invasive disease at a large tertiary institute in Singapore from 2000 to 2007 were serotyped, with 84 (43.8 %) and 159 (82.8 %) isolates belonging to serotypes covered by the pneumococcal heptavalent conjugate and polysaccharide vaccines, respectively. All non-meningitis isolates were susceptible to penicillin, and the attributable mortality was 21.4 %. Patients who fulfilled the US Advisory Committee on Immunization Practices criteria for vaccination with the pneumococcal polysaccharide vaccine comprised 74.0 % of the study cohort and had a significantly higher mortality risk.

Simplified large-scale Sanger genome sequencing for influenza A/H3N2 virus.

BACKGROUND: The advent of next-generation sequencing technologies and the resultant lower costs of sequencing have enabled production of massive amounts of data, including the generation of full genome sequences of pathogens. However, the small genome size of the influenza virus arguably justifies the use of the more conventional Sanger sequencing technology which is still currently more readily available in most diagnostic laboratories. RESULTS: We present a simplified Sanger-based genome sequencing method for sequencing the influenza A/H3N2 virus in a large-scale format. The entire genome sequencing was completed with 19 reverse transcription-polymerase chain reactions (RT-PCRs) and 39 sequencing reactions. This method was tested on 15 native clinical samples and 15 culture isolates, respectively, collected between 2009 and 2011. The 15 native clinical samples registered quantification cycle values ranging from 21.0 to 30.56, which were equivalent to 2.4×10(3)-1.4×10(6) viral copies/µL of RNA extract. All the PCR-amplified products were sequenced directly without PCR product purification. Notably, high quality sequencing data up to 700 bp were generated for all the samples tested. The completed sequence covered 408,810 nucleotides in total, with 13,627 nucleotides per genome, attaining 100% coding completeness. Of all the bases produced, an average of 89.49% were Phred quality value 40 (QV40) bases (representing an accuracy of circa one miscall for every 10,000 bases) or higher, and an average of 93.46% were QV30 bases (one miscall every 1000 bases) or higher. CONCLUSIONS: This sequencing protocol has been shown to be cost-effective and less labor-intensive in obtaining full influenza genomes. The constant high quality of sequences generated imparts confidence in extending the application of this non-purified amplicon sequencing approach to other gene sequencing assays, with appropriate use of suitably designed primers.

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.