Hong Kai Lee

A diagnostic polymerase chain reaction assay for Zika virus.

Zika virus (ZIKV) is a mosquito‐borne flavivirus. Infection results in a dengue‐like illness with fever, headache, malaise, and a maculopapular rash. Nearly all cases are mild and self‐limiting but in 2007, a large outbreak of ZIKV was reported from the island of Yap (in Micronesia, northwest of Indonesia). Singapore is already endemic for dengue, and its impact on public health and economic burden is significant. Other dengue‐like infections (e.g., Chikungunya virus) are present. Yet only 10% of reported dengue cases have laboratory confirmation. The identification and control of other dengue‐like, mosquito‐transmitted infections is thus important for the health of Singapores population, as well as its economy. Given that ZIKV shares the same Aedes mosquito vector with both dengue and Chikungunya, it is possible that this virus is present in Singapore and causing some of the mild dengue‐like illness. A specific and sensitive one‐step, reverse transcription polymerase chain reaction (RT‐PCR) with an internal control (IC) was designed and tested on 88 archived samples of dengue‐negative, Chikungunya‐negative sera from patients presenting to our hospital with a dengue‐like illness, to determine the presence of ZIKV in Singapore. The assay was specific for detection of ZIKV and displayed a lower limit of detection (LoD) of 140 copies viral RNA/reaction when tested on synthetic RNA standards prepared using pooled negative patient plasma. Of the 88 samples tested, none were positive for ZIKV RNA, however, the vast majority of these were from patients admitted to hospital and further study may be warranted in community‐based environments. J. Med. Virol. 84:1501–1505, 2012.

Differing Symptom Patterns in Early Pandemic vs Seasonal Influenza Infections

BACKGROUND Singapore is a tropical country with a temperature range of 23 degrees C to 35 degrees C and relative humidity of 48% to 100% throughout the year. Influenza incidence peaks in June through July and November through January, though influenza cases can be detected throughout the year. METHODS Between May 1 and July 28, 2009, a novel dual-gene diagnostic polymerase chain reaction assay targeting the hemagglutinin (HA) and nucleoprotein (NP) genes of the new influenza A(H1N1/2009) virus was specifically designed for enhanced influenza surveillance using nasopharyngeal swabs collected from symptomatic patients (including their close contacts) and returning travelers returning from influenza A(H1N1/2009)-affected areas, presenting to affiliated primary care clinics as well as the main hospital emergency department. RESULTS From the week of June 16 to June 23, 2009, this pandemic influenza A(H1N1/2009) displaced and then replaced the seasonal influenzas (H3N2, H1N1, and B). Of 2683 samples tested during this 12-week surveillance period, 742 (27.6%) were positive for any influenza virus using this assay, with 547 cases of A(H1N1/2009) (20.4%), 167 cases of A(H3N2) (6.2%), 14 cases of A(H1N1) (0.5%), and 12 cases of influenza B (0.4%). Results of multivariate analysis showed that age (P < .001), fever (P < .001), cough (P < .001), sore throat (P = .002), rhinorrhea (P = .001), and dyspnea (P < .001) were significantly different among these groups. CONCLUSIONS From this large prospective study, there was a lower incidence of fever and dyspnea in patients with pandemic influenza A(H1N1/2009) infection. Similar to reports from elsewhere, it was also found that this pandemic virus tends to infect younger people, though with fewer symptoms, on average, than seasonal influenza. Early pandemic influenza A(H1N1/2009) infections appeared to be slightly milder than seasonal influenza as indicated by different symptom patterns in the presentation of more than 500 cases of influenza A(H1N1/2009) during April through July to a large teaching hospital in Singapore.

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.

Diagnostic Testing for Pandemic Influenza in Singapore: A Novel Dual-Gene Quantitative Real-Time RT-PCR for the Detection of Influenza A/H1N1/2009

With the relative global lack of immunity to the pandemic influenza A/H1N1/2009 virus that emerged in April 2009 as well as the sustained susceptibility to infection, rapid and accurate diagnostic assays are essential to detect this novel influenza A variant. Among the molecular diagnostic methods that have been developed to date, most are in tandem monoplex assays targeting either different regions of a single viral gene segment or different viral gene segments. We describe a dual-gene (duplex) quantitative real-time RT-PCR method selectively targeting pandemic influenza A/H1N1/2009. The assay design includes a primer-probe set specific to only the hemagglutinin (HA) gene of this novel influenza A variant and a second set capable of detecting the nucleoprotein (NP) gene of all swine-origin influenza A virus. In silico analysis of the specific HA oligonucleotide sequence used in the assay showed that it targeted only the swine-origin pandemic strain; there was also no cross-reactivity against a wide spectrum of noninfluenza respiratory viruses. The assay has a diagnostic sensitivity and specificity of 97.7% and 100%, respectively, a lower detection limit of 50 viral gene copies/PCR, and can be adapted to either a qualitative or quantitative mode. It was first applied to 3512 patients with influenza-like illnesses at a tertiary hospital in Singapore, during the containment phase of the pandemic (May to July 2009).

Global epidemiology of non-influenza RNA respiratory viruses: data gaps and a growing need for surveillance

Summary Together with influenza, the non-influenza RNA respiratory viruses (NIRVs), which include respiratory syncytial virus, parainfluenza viruses, coronavirus, rhinovirus, and human metapneumovirus, represent a considerable global health burden, as recognised by WHOs Battle against Respiratory Viruses initiative. By contrast with influenza viruses, little is known about the contemporaneous global diversity of these viruses, and the relevance of such for development of pharmaceutical interventions. Although far less advanced than for influenza, antiviral drugs and vaccines are in different stages of development for several of these viruses, but no interventions have been licensed. This scarcity of global genetic data represents a substantial knowledge gap and impediment to the eventual licensing of new antiviral drugs and vaccines for NIRVs. Enhanced genetic surveillance will assist and boost research and development into new antiviral drugs and vaccines for these viruses. Additionally, understanding the global diversity of respiratory viruses is also part of emerging disease preparedness, because non-human coronaviruses and paramyxoviruses have been listed as priority concerns in a recent WHO research and development blueprint initiative for emerging infectious diseases. In this Personal View, we explain further the rationale for expanding the genetic database of NIRVs and emphasise the need for greater investment in this area of research.

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 2.4×103–1.4×106 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.

A Universal Influenza A and B Duplex Real-time RT-PCR Assay

A high throughput universal influenza A and B duplex real‐time RT‐PCR was developed to meet effectively the heightened surveillance and diagnostic needs essential in managing influenza infections and outbreaks. Primers and probes, designed to target highly conserved regions of the matrix protein of influenza A and the nucleoprotein of influenza B, were optimized using the high‐throughput LightCycler 480 II system. Analytical sensitivity and specificity were characterized using RNA transcripts diluted serially, archived non‐influenza respiratory viruses, and proficiency test samples. Eighty‐nine clinical samples were tested in parallel against existing influenza A and B monoplex assays. Once validated, the duplex assay was applied prospectively on 2,458 clinical specimens that were later subtyped. In April 2011, the emergence of an influenza B variant necessitated the inclusion of an additional modified probe for influenza B and revalidation of the revised protocol. The lower detection limits of the assay were 50 copies/PCR. There was no cross‐reactivity against any non‐influenza respiratory virus and all proficiency testing materials were identified correctly. The parallel testing revealed a 98.9% overall agreement. Routine application of the assay revealed high sensitivity and specificity for the detection of influenza A/H1N1/2009, A/H3N2 and influenza B. Assay Cq values correlated well between the pre‐ and post‐revision protocols for influenza A (r2 = 0.998) and B (r2 = 0.999). The revised protocol detected three additional novel influenza B variant cases in 200 specimens reported previously as influenza B negative. This in‐house assay offers a highly sensitive and specific option for laboratories seeking to expand their influenza testing capacity. J. Med. Virol. 84:1646–1651, 2012.

High-Resolution Melting Approach to Efficient Identification and Quantification of H275Y Mutant Influenza H1N1/2009 Virus in Mixed-Virus-Population Samples

ABSTRACT The single-nucleotide variation 823C to T (His275Tyr), responsible for oseltamivir drug resistance has been detected in some isolates of the influenza A/H1N1/2009 virus. Early detection of the presence of this oseltamivir-resistant strain allows prompt consideration of alternative treatment options. An isolated-probe–asymmetric amplification PCR (Roche LightCycler v2.0) and high-resolution melting (HRM) method using unlabeled probes and amplified products (Idaho LightScanner 32) was designed and optimized to detect and estimate the proportion of H275Y mutants in influenza A/H1N1/2009 virus samples. The lower limit of quantification within the linear range of PCR assay detection was 200 copies/reaction. The melting peaks of the H275Y-specific unlabeled probe for the wild-type A/H1N1/2009 and H275Y mutant viruses were clearly distinguishable at 65.5°C and 69.0°C, respectively, at various ratios of wild-type/mutant virus population standards. The 95% detection limit for the 10% mutant sample pool was 1,200 copies/reaction (95% confidence interval, 669.7 to 3,032.6 copies/reaction). This HRM assay was tested with 116 archived clinical specimens. The quantitative HRM results obtained with samples containing mixed mutant–wild-type virus populations, at threshold cycle (CT ) values of <29, compared well to those obtained with a pyrosequencing method performed by an independent laboratory. The quantitative feature of this assay allows the proportions of mutant and wild-type viral populations to be determined, which may assist in the conventional clinical management of infected patients and potentially more preemptive clinical management. This validated quantitative HRM method, with its low running cost, is well positioned as a rapid, high-throughput screening tool for oseltamivir resistance mutations in influenza A/H1N1/2009 virus-infected patients, with the potential to be adapted to other influenza virus species.

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.

Mixtures of oseltamivir-sensitive and -resistant pandemic influenza A/H1N1/2009 viruses in immunocompromised hospitalized children.

We report on 3 immunocompromised children infected with pandemic influenza A/H1N1/2009 in whom mixtures of oseltamivir-susceptible and oseltamivir-resistant viral populations developed, despite them receiving relatively short-term courses of oseltamivir. In addition, it was found that bacterial coinfections were common, indicating that empiric, antibiotics should be considered in such patients when infected with influenza virus.