Lynette Oon

Analysis of deaths during the severe acute respiratory syndrome (SARS) epidemic in Singapore: challenges in determining a SARS diagnosis.

CONTEXT An outbreak of severe acute respiratory syndrome (SARS), an infectious disease attributed to a novel coronavirus, occurred in Singapore during the first quarter of 2003 and led to 204 patients with diagnosed illnesses and 26 deaths by May 2, 2003. Twenty-one percent of these patients required admission to the medical intensive care unit. During this period, the Center for Forensic Medicine, Health Sciences Authority, Singapore, performed a total of 14 postmortem examinations for probable and suspected SARS. Of these, a total of 8 were later confirmed as SARS infections. OBJECTIVE Our series documents the difficulties encountered at autopsy during the initial phases of the SARS epidemic, when the pattern of infection and definitive diagnostic laboratory criteria were yet to be established. DESIGN Autopsies were performed by pathologists affiliated with the Center for Forensic Medicine, Health Sciences Authority, Singapore. Tissue was accessed and read at the Tan Tock Seng Hospital, Singapore, and at the Armed Forces Institute of Pathology, Washington, DC. Autopsy tissue was submitted to the Virology Department, Singapore General Hospital, for analysis, and in situ hybridization for the SARS coronavirus was carried out at the National Institute of Infectious Diseases, Tokyo, Japan. RESULTS Thirteen of 14 patients showed features of diffuse alveolar damage. In 8 patients, no precipitating etiology was identified, and in all of these patients, we now have laboratory confirmation of coronavirus infection. Two of the 8 patients presented at autopsy as sudden unexpected deaths, while the remaining 6 patients had been hospitalized with varying lengths of stay in the intensive care unit. In 3 patients, including the 2 sudden unexpected deaths, in situ hybridization showed the presence of virally infected cells within the lung. In 4 of the 8 SARS patients, pulmonary thromboemboli were also recognized on gross examination, while one patient had marantic cardiac valvular vegetations. CONCLUSIONS It is unfortunate that the term atypical pneumonia has been used in conjunction with SARS. Although nonspecific by itself, the term does not accurately reflect the underlying dangers of viral pneumonia, which may progress rapidly to acute respiratory distress syndrome. We observed that the clinical spectrum of disease as seen in our autopsy series included sudden deaths. This is a worrisome finding that illustrates that viral diseases will have a spectrum of clinical presentations and that the diagnoses made for such patients must incorporate laboratory as well as clinical data.

Pandemic (H1N1) 2009 infection in adult solid organ transplant recipients in Singapore.

Background. Influenza can produce significant complications in immunocompromised persons. Methods. We studied the effects of the pandemic (H1N1) 2009 (pH1N1) infection on solid organ transplant recipients in our hospital, with emphasis on clinical information, duration of viral culture positivity, polymerase chain reaction positivity, effects of oseltamivir therapy, and graft status at 6 months of follow-up. Results. Twenty-two cases of pH1N1 infection involving 18 renal, two lung, one heart, and one liver transplant recipients were seen from July 14 to September 8, 2009. Their median age was 50.5 years (range 20-70 years); 64% were women, and median time posttransplant was 40 months (range 6-204 months). Common symptoms were fever (86%), cough (77%), sore throat (55%), phlegm (32%), and myalgia (27%). The median duration of symptoms (n=21) and duration of polymerase chain reaction positivity (n=15) were 7 (range 4-13 days) and 8 days (range 4-16 days), respectively. Mean (±SD) duration of symptom resolution (7.4±3.0 vs. 7.8±3.0 days, P=0.76) and viral culture positivity (5.3±2.8 vs. 4.3±3.2 days, P=0.65) did not differ between those who received a 5-day (n=9) or 10-day (n=12) course of oseltamivir. Five patients (22.7%) developed pneumonia with three needing intensive care. Mortality rate was 4.5% (1/22). At 6 months, three graft rejections involving two renal and one lung developed. Conclusions. Our findings indicate that the pH1N1 infection in solid organ transplant recipients is associated with some degree of morbidity and may affect the function of the transplanted organ. In this nonrandomized comparison, patients treated with 5 days of oseltamivir did not fare worse compared with those who received 10 days.

Comparison of Circulating Tumour Cells and Circulating Cell-Free Epstein-Barr Virus DNA in Patients with Nasopharyngeal Carcinoma Undergoing Radiotherapy

Quantification of Epstein-Barr virus (EBV) cell-free DNA (cfDNA) is commonly used in clinical settings as a circulating biomarker in nasopharyngeal carcinoma (NPC), but there has been no comparison with circulating tumour cells (CTCs). Our study aims to compare the performance of CTC enumeration against EBV cfDNA quantitation through digital PCR (dPCR) and quantitative PCR. 74 plasma samples from 46 NPC patients at baseline and one month after radiotherapy with or without concurrent chemotherapy were analysed. CTCs were captured by microsieve technology and enumerated, while three different methods of EBV cfDNA quantification were applied, including an in-house qPCR assay for BamHI-W fragment, a CE-IVD qPCR assay (Sentosa ®) and a dPCR (Clarity™) assay for Epstein-Barr nuclear antigen 1 (EBNA1). EBV cfDNA quantitation by all workflows showed stronger correlation with clinical stage, radiological response and overall survival in comparison with CTC enumeration. The highest detection rate of EBV cfDNA in pre-treatment samples was seen with the BamHI-W qPCR assay (89%), followed by EBNA1-dPCR (85%) and EBNA1-qPCR (67%) assays. Overall, we show that EBV cfDNA outperforms CTC enumeration in correlation with clinical outcomes of NPC patients undergoing treatment. Techniques such as dPCR and target selection of BamHI-W may improve sensitivity for EBV cfDNA detection.

Phylodynamics of H1N1/2009 influenza reveals the transition from host adaptation to immune-driven selection

Influenza A H1N1/2009 virus that emerged from swine rapidly replaced the previous seasonal H1N1 virus. Although the early emergence and diversification of H1N1/2009 is well characterized, the ongoing evolutionary and global transmission dynamics of the virus remain poorly investigated. To address this we analyse >3,000 H1N1/2009 genomes, including 214 full genomes generated from our surveillance in Singapore, in conjunction with antigenic data. Here we show that natural selection acting on H1N1/2009 directly after introduction into humans was driven by adaptation to the new host. Since then, selection has been driven by immunological escape, with these changes corresponding to restricted antigenic diversity in the virus population. We also show that H1N1/2009 viruses have been subject to regular seasonal bottlenecks and a global reduction in antigenic and genetic diversity in 2014.

Molecular epidemiology of norovirus in Singapore, 2004–2011

Norovirus (NoV) is the most common cause of sporadic and epidemic gastroenteritis, globally. This study aimed to investigate the molecular epidemiology of NoV‐associated acute gastroenteritis in Singapore by classifying circulating NoV genotypes and genogroup II, genotype 4 (GII.4) variants between September 2004 and February 2011. The temporal dominance and antigenic variation within the circulating epidemic NoV GII.4 variants was also examined, in order to compare the trends in Singapore to those observed globally during the same period. A total of 312 of 1,060 fecal specimens were positive for NoV RNA, using a quantitative RT‐PCR. In a subset (125 of 312) of NoV positive samples, the 5′ end of ORF2 (region C) of the GI or GII NoV genome was amplified and sequenced. Subsequent phylogenetic analysis identified GII.4 was the most commonly identified genotype representing 80.8% (101/125) of NoV sequenced in this study. The predominant GII.4 variants in circulation during the 2004–2011 epidemic periods were Hunter 2004 (2004–2005), Den Haag 2006b (2006–2009), and New Orleans 2009 (2009–2011). Amino acid variation within the P2 domain of the major capsid protein, VP1, was followed longitudinally within the GII.4 lineage. A constant turnover of variant‐specific amino acid change was observed, particularly within the antigenic epitopes A, C and E. In conclusion, this study has characterized the NoV strains in circulation in Singapore between 2004 and 2011. The molecular epidemiology and persistence of GII.4 pandemic NoV lineages in Singapore was similar to trends seen globally, with a noted absence of the Asia 2003 variant. J Med. Virol. 85:1842–1851, 2013.

Atypical SARS and Escherichia coli Bacteremia

We describe a patient with severe acute respiratory syndrome (SARS) whose clinical symptoms were masked by Escherichia coli bacteremia. SARS developed in a cluster of healthcare workers who had contact with this patient. SARS was diagnosed when a chest infiltrate developed and when the patient’s brother was hospitalized with acute respiratory failure. We highlight problems in atypical cases and offer infection control suggestions.

Prevalence of cervical human papillomavirus infection in healthy women is related to sexual behaviours and educational level: a cross-sectional study

This study reports the prevalence and risk factors of human papillomavirus (HPV) infection in healthy women in Singapore. Demography, education, sexual and reproductive history and cigarette smoking habits were obtained from a cross-sectional population of healthy women and girls aged above 12 years of age. Cervical or vaginal cytology samples were investigated for 37 known anogenital HPV subtypes using the linear array PCR method. Chi square statistics were used to test for associations of individual epidemiological factors with HPV infection. Independent risk factors were identified with binomial logistic regression analysis. Of 891 subjects, the prevalence of HPV infection was 9.31% (83/891 women) for any-type HPV and 5.05% (46/891 women) for the high-risk HPV (hrHPV). Of 30 HPV subtypes detected, the most prevalent genotypes in descending order of frequency were subtypes 51, 16, 52, 58 and 66 for hrHPV and subtypes 62, 61, 84, 72 and 53 for the low-risk HPV. This frequency distribution of HPV subtypes was different from reports from other countries within Asia. Forty-six virgins studied tested negative for HPV infection. Significant independent risk factors for any-type HPV infection were multiple sexual partners (adjusted OR 1.4) and low (≤6 years) educational level (adjusted OR 4.0). The distribution of HPV subtypes in healthy women varies between different countries within Asia. In Singapore, the prevalence of HPV infection was 9.31% and was related to penetrative sexual intercourse, multiple sexual partners and low educational level.

Impact of Smoking and Brain Metastasis on Outcomes of Advanced EGFR Mutation Lung Adenocarcinoma Patients Treated with First Line Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors.

Objectives This purpose of this study was to examine clinical-pathologic factors – particularly smoking and brain metastases – in EGFR mutation positive (M+) lung adenocarcinoma (ADC) to determine their impact on survival in patients treated with first line EGFR TKI. Methods A retrospective review of EGFR mutation reflex testing experience for all ADC diagnosed at a tertiary Asian cancer centre from January 2009 to April 2013. Amongst this cohort, patients with advanced EGFR M+ ADC treated with first line EGFR TKI were identified to determine factors that influence progression free and overall survival. Results 444/742 (59.8%) ADC reflex tested for EGFR mutations were EGFR M+. Amongst never-smokers (n=468), EGFR M+ were found in 74.5% of females and 76.3% of males, and amongst ever smokers (n=283), in 53.3% of females and 35.6% of males. Exon 20 mutations were found more commonly amongst heavy smokers (> 50 pack years and > 20 pack years, Pearson’s chi square p=0.044, and p=0.038 respectively). 211 patients treated with palliative first line TKI had a median PFS and OS of 9.2 and 19.6 months respectively. 26% of patients had brain metastasis at diagnosis. This was significantly detrimental to overall survival (HR 1.85, CI 1.09-3.16, p=0.024) on multivariate analysis. There was no evidence that smoking status had a significant impact on survival. Conclusions The high prevalence of EGFR M+ in our patient population warrants reflex testing regardless of gender and smoking status. Smoking status and dosage did not impact progression free or overall survival in patients treated with first line EGFR TKI. The presence of brain metastasis at diagnosis negatively impacts overall survival.

Arginine catabolic mobile element in methicillin-resistant Staphylococcus aureus (MRSA) clonal group ST239-MRSA-III isolates in Singapore: implications for PCR-based screening tests.

Espedido and coworkers found that up to 38.7% of methicillin-resistant Staphylococcus aureus (MRSA) clonal group ST239-MRSA-III strains in a Sydney hospital carried an arginine catabolic mobile element (ACME) type II variant that inserted between orfX and the staphylococcal cassette chromosome mec

Gastrointestinal colonisation of vancomycin-resistant enterococcus in a singapore teaching hospital

Summary Vancomycin‐resistant enterococcus (VRE) has become an important nosocomial pathogen in many countries but is still rare in Singapore. A study was conducted from January to March 1997 at a 900‐bed teaching hospital to determine the prevalence of intestinal colonisation of VRE in the patient population. A total of 299 consecutive stool specimens received by the microbiology laboratory for routine testing were plated onto two different selective media for comparison. VRE isolated were phenotypically characterised using minimum inhibitory concentrations (MICs) to vancomycin and teicoplanin and then typed using pulsed‐field gel electrophoresis (PFGE) with Sma I digestion of DNA. VRE were detected in the stool of 35 patients (12.3%). This group consisted of four isolates with VanB (one Enterococcus faecalis and three E. faecium ) and 31 isolates with VanC (30 E. casseliflavus and one E. gallinarum). Two patients (0.7%) carried isolates (both VanB) with high level resistance to vancomycin (MIC ≥ 256 μg/ml) while the rest had isolates of low level resistance (MIC = 8 μg/ml). Except for isolates from the same patients, PFGE patterns were diverse, suggesting that the VRE isolates were genotypically different and possibly introduced from many sources. This study demonstrates that VRE colonisation is not uncommon in the Singapore patient population.Abbreviations: BHIA, brain heart infusion agar; HLAR, high‐level aminoglycoside resistance; MHA, Mueller‐Hinton agar; MIC, inimum inhibitory concentration; NCCLS, National Committee for Clinical Laboratory Standards; PFGE, pulsed‐field gel electrophoresis; QC, quality control; VRE, vancomycin‐resistant enterococcus.