Divergent evolutionary trajectories of influenza B viruses underlie their contemporaneous epidemic activity.

Influenza B viruses have circulated in humans for over 80 y, causing a significant disease burden. Two antigenically distinct lineages ("B/Victoria/2/87-like" and "B/Yamagata/16/88-like," termed Victoria and Yamagata) emerged in the 1970s and have cocirculated since 2001. Since 2015 both lineages have shown unusually high levels of epidemic activity, the reasons for which are unclear. By analyzing over 12,000 influenza B virus genomes, we describe the processes enabling the long-term success and recent resurgence of epidemics due to influenza B virus. We show that following prolonged diversification, both lineages underwent selective sweeps across the genome and have subsequently taken alternate evolutionary trajectories to exhibit epidemic dominance, with no reassortment between lineages. Hemagglutinin deletion variants emerged concomitantly in multiple Victoria virus clades and persisted through epistatic mutations and interclade reassortment-a phenomenon previously only observed in the 1970s when Victoria and Yamagata lineages emerged. For Yamagata viruses, antigenic drift of neuraminidase was a major driver of epidemic activity, indicating that neuraminidase-based vaccines and cross-reactivity assays should be employed to monitor and develop robust protection against influenza B morbidity and mortality. Overall, we show that long-term diversification and infrequent selective sweeps, coupled with the reemergence of hemagglutinin deletion variants and antigenic drift of neuraminidase, are factors that contributed to successful circulation of diverse influenza B clades. Further divergence of hemagglutinin variants with poor cross-reactivity could potentially lead to circulation of 3 or more distinct influenza B viruses, further complicating influenza vaccine formulation and highlighting the urgent need for universal influenza vaccines.

Molecular insights into evolution, mutations and receptor-binding specificity of influenza A and B viruses from outpatients and hospitalized patients in Singapore.

BACKGROUND: This study compared the genomes of influenza viruses that caused mild infections among outpatients and severe infections among hospitalized patients in Singapore, and characterized their molecular evolution and receptor-binding specificity. METHODS: The complete genomes of influenza A/H1N1, A/H3N2 and B viruses that caused mild infections among outpatients and severe infections among inpatients in Singapore during 2012-2015 were sequenced and characterized. Using various bioinformatics approaches, we elucidated their evolutionary, mutational and structural patterns against the background of global and vaccine strains. RESULTS: The phylogenetic trees of the 8 gene segments revealed that the outpatient and inpatient strains overlapped with representative global and vaccine strains. We observed a cluster of inpatients with A/H3N2 strains that were closely related to vaccine strain A/Texas/50/2012(H3N2). Several protein sites could accurately discriminate between outpatient versus inpatient strains, with site 221 in neuraminidase (NA) achieving the highest accuracy for A/H3N2. Interestingly, amino acid residues of inpatient but not outpatient isolates at those sites generally matched the corresponding residues in vaccine strains, except at site 145 of hemagglutinin (HA). This would be especially relevant for future surveillance of A/H3N2 strains in relation to their antigenicity and virulence. Furthermore, we observed a trend in which the HA proteins of influenza A/H3N2 and A/H1N1 exhibited enhanced ability to bind both avian and human host cell receptors. In contrast, the binding ability to each receptor was relatively stable for the HA of influenza B. CONCLUSIONS: Overall, our findings extend our understanding of the molecular and structural evolution of influenza virus strains in Singapore within the global context of these dynamic viruses.

High Yield of HIV Testing in Dengue-Like Febrile Illness in Singapore.

Where dengue virus infections are endemic, acute febrile illness is often managed as dengue fever (DF) without diagnostic testing. In a prospective study of 140 patients with clinical features of DF, 3 (2.1%) had acute HIV infection (AHI). We recommend testing for AHI in dengue-like febrile illness.

High Viral Diversity and Mixed Infections in Cerebral Spinal Fluid From Cases of Varicella Zoster Virus Encephalitis.

Background: Varicella zoster virus (VZV) may cause encephalitis, both with and without rash. Here we investigate whether viruses recovered from the central nervous system (CNS; encephalitis or meningitis) differ genetically from those recovered from non-CNS samples. Methods: Enrichment-based deep sequencing of 45 VZV genomes from cerebral spinal fluid (CSF), plasma, bronchoalveolar lavage (BAL), and vesicles was carried out with samples collected from 34 patients with and without VZV infection of the CNS. Results: Viral sequences from multiple sites in the same patient were identical at the consensus level. Virus from vesicle fluid and CSF in cases of meningitis showed low-level diversity. By contrast, plasma, BAL, and encephalitis had higher numbers of variant alleles. Two CSF-encephalitis samples had high genetic diversity, with variant frequency patterns typical of mixed infections with different clades. Conclusions: Low viral genetic diversity in vesicle fluid is compatible with previous observations that VZV skin lesions arise from single or low numbers of virions. A similar result was observed in VZV from cases of VZV meningitis, a generally self-limiting infection. CSF from cases of encephalitis had higher diversity with evidence for mixed clade infections in 2 cases. We hypothesize that reactivation from multiple neurons may contribute to the pathogenesis of VZV encephalitis.

Longitudinal monitoring reveals dynamic changes in circulating tumor cells (CTCs) and CTC-associated miRNAs in response to chemotherapy in metastatic colorectal cancer patients.

We evaluated the changes in CTC count and CTC-associated miRNAs during the course of chemotherapy in patients with metastatic colorectal cancer. Blood samples were collected from 9 metastatic colorectal cancer patients prior to chemotherapy and at every other chemotherapy session during the course of treatment. CTCs were isolated and enumerated using a size-exclusion method (CellSievo, Singapore). CTC-associated miRNAs were isolated using a paper-based, partitioning method, and analyzed using reverse transcription quantitative real-time PCR (MiRXES, Singapore). CTC count trends generally correlated with disease progression defined by radiological measurements and trends in carcinoembryonic antigen (CEA) levels; hence CTC counts may be useful in cases where CEA is not elevated. CTC-associated miRNAs identified were miR-15b, miR-16, miR-19a, miR-21, miR-25, miR-30d, miR-126, miR-185, miR-221, miR-222, and miR-324-5p. The expression of CTC-associated miRNAs did not appear to correlate with CTC count and exhibited inter-individual heterogeneity. This pilot study suggests that analysis of CTC changes during the course of treatment may be useful in monitoring response to therapy in metastatic colorectal cancer.

Impact of Outpatient Neuraminidase Inhibitor Treatment in Patients Infected With Influenza A(H1N1)pdm09 at High Risk of Hospitalization: An Individual Participant Data Metaanalysis.

BACKGROUND: While evidence exists to support the effectiveness of neuraminidase inhibitors (NAIs) in reducing mortality when given to hospitalized patients with A(H1N1)pdm09 virus infection, the impact of outpatient treatment on hospitalization has not been clearly established. We investigated the impact of outpatient NAI treatment on subsequent hospitalization in patients with A(H1N1)pdm09 virus infection. METHODS: We assembled general community and outpatient data from 9 clinical centers in different countries collected between January 2009 and December 2010. We standardized data from each study center to create a pooled dataset and then used mixed-effects logistic regression modeling to determine the effect of NAI treatment on hospitalization. We adjusted for NAI treatment propensity and preadmission antibiotic use, including "study center" as a random intercept to account for differences in baseline hospitalization rate between centers. RESULTS: We included 3376 patients with influenza A(H1N1)pdm09, of whom 3085 (91.4%) had laboratory-confirmed infection. Eight hundred seventy-three patients (25.8%) received outpatient or community-based NAI treatment, 928 of 2395 (38.8%) with available data had dyspnea or respiratory distress, and hospitalizations occurred in 1705 (50.5%). After adjustment for preadmission antibiotics and NAI treatment propensity, preadmission NAI treatment was associated with decreased odds of hospital admission compared to no NAI treatment (adjusted odds ratio, 0.24; 95% confidence interval, 0.20-0.30). CONCLUSIONS: In a population with confirmed or suspected A(H1N1)pdm09 and at high risk of hospitalization, outpatient or community-based NAI treatment significantly reduced the likelihood of requiring hospital admission. These data suggest that community patients with severe influenza should receive NAI treatment.

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.

Sampling circulating tumor cells for clinical benefits: how frequent?

Circulating tumor cells (CTCs) are cells shed from tumors or metastatic sites and are a potential biomarker for cancer diagnosis, management, and prognostication. The majority of current studies use single or infrequent CTC sampling points. This strategy assumes that changes in CTC number, as well as phenotypic and molecular characteristics, are gradual with time. In reality, little is known today about the actual kinetics of CTC dissemination and phenotypic and molecular changes in the blood of cancer patients. Herein, we show, using clinical case studies and hypothetical simulation models, how sub-optimal CTC sampling may result in misleading observations with clinical consequences, by missing out on significant CTC spikes that occur in between sampling times. Initial studies using highly frequent CTC sampling are necessary to understand the dynamics of CTC dissemination and phenotypic and molecular changes in the blood of cancer patients. Such an improved understanding will enable an optimal, study-specific sampling frequency to be assigned to individual research studies and clinical trials and better inform practical clinical decisions on cancer management strategies for patient benefits.

Recombination of Globally Circulating Varicella-Zoster Virus.

UNLABELLED: Varicella-zoster virus (VZV) is a human herpesvirus, which during primary infection typically causes varicella (chicken pox) and establishes lifelong latency in sensory and autonomic ganglia. Later in life, the virus may reactivate to cause herpes zoster (HZ; also known as shingles). To prevent these diseases, a live-attenuated heterogeneous vaccine preparation, vOka, is used routinely in many countries worldwide. Recent studies of another alphaherpes virus, infectious laryngotracheitis virus, demonstrate that live-attenuated vaccine strains can recombine in vivo, creating virulent progeny. These findings raised concerns about using attenuated herpesvirus vaccines under conditions that favor recombination. To investigate whether VZV may undergo recombination, which is a prerequisite for VZV vaccination to create such conditions, we here analyzed 115 complete VZV genomes. Our results demonstrate that recombination occurs frequently for VZV. It thus seems that VZV is fully capable of recombination if given the opportunity, which may have important implications for continued VZV vaccination. Although no interclade vaccine wild-type recombinant strains were found, intraclade recombinants were frequently detected in clade 2, which harbors the vaccine strains, suggesting that the vaccine strains have already been involved in recombination events, either in vivo or in vitro during passages in cell culture. Finally, previous partial and complete genomic studies have described strains that do not cluster phylogenetically to any of the five established clades. The additional VZV strains sequenced here, in combination with those previously published, have enabled us to formally define a novel sixth VZV clade. IMPORTANCE: Although genetic recombination has been demonstrated to frequently occur for other human alphaherpesviruses, herpes simplex viruses 1 and 2, only a few ancient and isolated recent recombination events have hitherto been demonstrated for VZV. In the present study, we demonstrate that VZV also frequently undergoes genetic recombination, including strains belonging to the clade containing the vOKA strain.

Bitten by a bug or a bag? Transfusion-transmitted dengue: a rare complication in the bleeding surgical patient.

BACKGROUND: Blood-borne infections remain a risk of blood transfusions. While routine screening of donated blood products has greatly reduced the risk of human immunodeficiency virus, hepatitis B, and hepatitis C transmission, arboviruses such as dengue, chikungunya, and the West Nile virus remain significant risks especially during outbreaks. CASE REPORT: We report a rare case of dengue documented to be acquired through a blood transfusion, which resulted in severe thrombocytopenia prolonging admission in hospital in a neurosurgical patient. RESULTS: The donor of one of the units of red blood cells presented with dengue fever 2 days after donating. Sanger sequencing confirmed DENV-2 (dengue virus, Serotype 2) in both the donor and the patient samples and showed 100% nucleotide sequence identity between the two viruses, confirming transfusion-transmitted dengue infection. CONCLUSION: This case highlights the importance of arboviral screening of donor blood, especially for populations in endemic areas during outbreaks.

Absence of detectable influenza RNA transmitted via aerosol during various human respiratory activities--experiments from Singapore and Hong Kong.

Two independent studies by two separate research teams (from Hong Kong and Singapore) failed to detect any influenza RNA landing on, or inhaled by, a life-like, human manikin target, after exposure to naturally influenza-infected volunteers. For the Hong Kong experiments, 9 influenza-infected volunteers were recruited to breathe, talk/count and cough, from 0.1 m and 0.5 m distance, onto a mouth-breathing manikin. Aerosolised droplets exhaled from the volunteers and entering the manikin's mouth were collected with PTFE filters and an aerosol sampler, in separate experiments. Virus detection was performed using an in-house influenza RNA reverse-transcription polymerase chain reaction (RT-PCR) assay. No influenza RNA was detected from any of the PTFE filters or air samples. For the Singapore experiments, 6 influenza-infected volunteers were asked to breathe (nasal/mouth breathing), talk (counting in English/second language), cough (from 1 m/0.1 m away) and laugh, onto a thermal, breathing manikin. The manikin's face was swabbed at specific points (around both eyes, the nostrils and the mouth) before and after exposure to each of these respiratory activities, and was cleaned between each activity with medical grade alcohol swabs. Shadowgraph imaging was used to record the generation of these respiratory aerosols from the infected volunteers and their impact onto the target manikin. No influenza RNA was detected from any of these swabs with either team's in-house diagnostic influenza assays. All the influenza-infected volunteers had diagnostic swabs taken at recruitment that confirmed influenza (A/H1, A/H3 or B) infection with high viral loads, ranging from 10(5)-10(8) copies/mL (Hong Kong volunteers/assay) and 10(4)-10(7) copies/mL influenza viral RNA (Singapore volunteers/assay). These findings suggest that influenza RNA may not be readily transmitted from naturally-infected human source to susceptible recipients via these natural respiratory activities, within these exposure time-frames. Various reasons are discussed in an attempt to explain these findings.

Rapid prenatal diagnosis of common beta-thalassemia mutations in Southeast Asia using pyrosequencing.

OBJECTIVE: Current methods of prenatal diagnosis to detect beta-thalassemia are Sanger sequencing and reverse dot blot. These methods are time-consuming and can prolong assay turnaround time. We aim to develop a sensitive and rapid method to detect 27 beta-thalassemia mutations using pyrosequencing. METHOD: Pyrosequencing primer pairs and sequencing primers were designed to detect 27 most common beta-thalassemia mutations found in Singapore. Pyrosequencing was performed on 191 DNA samples with known beta-thalassemia mutations isolated from 143 peripheral blood and 48 prenatal samples (seven chorionic villus biopsies, 26 cultured amniocytes, 15 uncultured amniocytes). All mutations were validated with Sanger sequencing. RESULTS: Pyrosequencing identified 210 alleles with beta-thalassemia mutations and 82 alleles without mutations with 100% sensitivity (lower 95% confidence interval [CI], 97.8%) and 100% specificity (lower 95% CI, 94.4%). All pyrosequences were concordant with Sanger-based sequences. Pyrosequencing was able to detect DNA concentrations as low as 2 ng, obviating the need for cell culture in volume-restricted samples. Sample receipt-to-report assay turnaround times were 16 to 18 h (Sanger sequencing) and 4 to 6 h (pyrosequencing). CONCLUSION: Pyrosequencing is a rapid and sensitive method to detect common beta-thalassemia mutations without the need for cell culture, thus reducing the assay turnaround time.

Two-step evolution of the hepatitis B drug-resistant mutations in a patient who developed primary entecavir resistance.

AIM: Few cases of primary entecavir resistance in chronic hepatitis B patients have been reported to date. The serial profiling of the HBV polymerase gene mutations from a treatment-naive patient who developed drug resistance after 32 months of entecavir therapy is presented here. DESIGN: Serum samples were collected at multiple time points from before the start of therapy to virological and biochemical breakthrough. The evolution of the hepatitis B virus polymerase gene mutations was analysed with commercial line probe assay and pyrosequencing. RESULTS: Drug resistance mutation analysis by pyrosequencing revealed a two-step process in the selection of drug resistance. The patient had a good initial response to entecavir 0.5 mg/day. A partially resistant HBV strain first emerged as the predominant species from as early as 2 weeks. After a period of non-compliance to therapy, there was virological breakthrough, which resolved on restarting entecavir. Shortly after, there was secondary failure of entecavir therapy, caused by a new resistant strain carrying all three mutations required. CONCLUSION: In this patient, pre-existence of minor population of partially resistant viral strains and treatment non-compliance probably contributed to his development of primary entecavir resistance.

Influenza outbreaks in Singapore: epidemiology, diagnosis, treatment and prevention.

With the recent influenza A/H1N1 2009 pandemic still spreading through global populations, there has been an increased focus on optimizing the prevention, diagnosis and treatment of influenza infections, as well as the epidemiology of the virus. Clinical and epidemiological data on influenza infections in tropical countries have been relatively sparse until fairly recently, and it is the aim of this review to close some of these gaps by examining the behavior of influenza viruses in the tropical Singaporean population.

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 Singapore's 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.

Angioimmunoblastic T-cell lymphoma with hyperplastic germinal centres (pattern 1) shows superior survival to patterns 2 and 3: a meta-analysis of 56 cases.

AIMS: Angioimmunoblastic T-cell lymphoma (AITL) may present in patterns 1, 2 or 3, representing those with hyperplastic, regressed or effaced germinal centres (GCs), respectively, but the prognostic utility of this subclassification has not been previously validated. METHODS AND RESULTS: Twenty-five cases of AITL were reviewed immunohistologically and with in-situ hybridization for Epstein-Barr virus-encoded RNA and polymerase chain reaction for T-cell receptor gamma and immunoglobulin heavy chain clonality and followed for up to 120 months. Four cases had conventional hyperplastic GCs, two had floral GCs, and one had progressively transformed GCs, consistent with pattern 1 and one additional case had hyalinized GCs, consistent with pattern 2. The remaining 17 (pattern 3) cases lacked morphologically discernible GCs. The Kaplan-Meier survival distribution of pattern 1 cases (5-year survival 83%) was superior to that of pattern 2 and 3 cases [5-year-survival 36% (P = 0.0417)] only when combined with the 31 cases, seven of which were pattern 1, that Attygalle et al. had followed for up to 247 months and previously published. Furthermore, the development of B-lineage (classical Hodgkin or diffuse large-cell) lymphoma was associated exclusively with pattern 3 (P = 0.0057). CONCLUSIONS: Pattern 1 represents an indolent phase/grade of AITL, unassociated with the development of secondary B-lineage lymphoma and uninfluenced by treatment regimen.

Plumbagin inhibits invasion and migration of breast and gastric cancer cells by downregulating the expression of chemokine receptor CXCR4.

BACKGROUND: Increasing evidence indicates that the interaction between the CXC chemokine receptor-4 (CXCR4) and its ligand CXCL12 is critical in the process of metastasis that accounts for more than 90% of cancer-related deaths. Thus, novel agents that can downregulate the CXCR4/CXCL12 axis have therapeutic potential in inhibiting cancer metastasis. METHODS: In this report, we investigated the potential of an agent, plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone), for its ability to modulate CXCR4 expression and function in various tumor cells using Western blot analysis, DNA binding assay, transient transfection, real time PCR analysis, chromatin immunoprecipitation, and cellular migration and invasion assays. RESULTS: We found that plumbagin downregulated the expression of CXCR4 in breast cancer cells irrespective of their HER2 status. The decrease in CXCR4 expression induced by plumbagin was not cell type-specific as the inhibition also occurred in gastric, lung, renal, oral, and hepatocellular tumor cell lines. Neither proteasome inhibition nor lysosomal stabilization had any effect on plumbagin-induced decrease in CXCR4 expression. Detailed study of the underlying molecular mechanism(s) revealed that the regulation of the downregulation of CXCR4 was at the transcriptional level, as indicated by downregulation of mRNA expression, inhibition of NF-κB activation, and suppression of chromatin immunoprecipitation activity. In addition, using a virtual, predictive, functional proteomics-based tumor pathway platform, we tested the hypothesis that NF-κB inhibition by plumbagin causes the decrease in CXCR4 and other metastatic genes. Suppression of CXCR4 expression by plumbagin was found to correlate with the inhibition of CXCL12-induced migration and invasion of both breast and gastric cancer cells. CONCLUSIONS: Overall, our results indicate, for the first time, that plumbagin is a novel blocker of CXCR4 expression and thus has the potential to suppress metastasis of cancer.

Comparison of pandemic (H1N1) 2009 and seasonal influenza viral loads, Singapore.

Mean viral loads for patients with pandemic (H1N1) 2009 were ≈1 log10 times lower than those for patients with seasonal influenza within the first week after symptom onset. Neither pandemic nor seasonal influenza viral loads correlated with clinical severity of illness. No correlation was found between viral loads and concurrent illness.