Unsupervised spatially embedded deep representation of spatial transcriptomics.

Optimal integration of transcriptomics data and associated spatial information is essential towards fully exploiting spatial transcriptomics to dissect tissue heterogeneity and map out inter-cellular communications. We present SEDR, which uses a deep autoencoder coupled with a masked self-supervised learning mechanism to construct a low-dimensional latent representation of gene expression, which is then simultaneously embedded with the corresponding spatial information through a variational graph autoencoder. SEDR achieved higher clustering performance on manually annotated 10 × Visium datasets and better scalability on high-resolution spatial transcriptomics datasets than existing methods. Additionally, we show SEDR's ability to impute and denoise gene expression (URL: https://github.com/JinmiaoChenLab/SEDR/ ).

Notch-dependent cooperativity between myeloid lineages promotes Langerhans cell histiocytosis pathology.

Langerhans cell histiocytosis (LCH) is a potentially fatal neoplasm characterized by the aberrant differentiation of mononuclear phagocytes, driven by mitogen-activated protein kinase (MAPK) pathway activation. LCH cells may trigger destructive pathology yet remain in a precarious state finely balanced between apoptosis and survival, supported by a unique inflammatory milieu. The interactions that maintain this state are not well known and may offer targets for intervention. Here, we used single-cell RNA-seq and protein analysis to dissect LCH lesions, assessing LCH cell heterogeneity and comparing LCH cells with normal mononuclear phagocytes within lesions. We found LCH discriminatory signatures pointing to senescence and escape from tumor immune surveillance. We also uncovered two major lineages of LCH with DC2- and DC3/monocyte-like phenotypes and validated them in multiple pathological tissue sites by high-content imaging. Receptor-ligand analyses and lineage tracing in vitro revealed Notch-dependent cooperativity between DC2 and DC3/monocyte lineages during expression of the pathognomonic LCH program. Our results present a convergent dual origin model of LCH with MAPK pathway activation occurring before fate commitment to DC2 and DC3/monocyte lineages and Notch-dependent cooperativity between lineages driving the development of LCH cells.

Uncoupling immune trajectories of response and adverse events from anti-PD-1 immunotherapy in hepatocellular carcinoma.

BACKGROUND & AIMS: While immune checkpoint blockade (ICB) has shown promise in patients with hepatocellular carcinoma (HCC), it is associated with modest response rates and immune-related adverse events (irAEs) are common. In this study, we aimed to decipher immune trajectories and mechanisms of response and/or irAEs in patients with HCC receiving anti-programmed cell death 1 (anti-PD-1) therapy. METHODS: Pre- and on-treatment peripheral blood samples (n = 60) obtained from 32 patients with HCC (Singapore cohort) were analysed by cytometry by time-of-flight and single-cell RNA sequencing, with flow cytometric validation in an independent Korean cohort (n = 29). Mechanistic validation was conducted by bulk RNA sequencing of 20 pre- and on-treatment tumour biopsies and using a murine HCC model treated with different immunotherapeutic combinations. RESULTS: Single-cell analyses identified CXCR3+CD8+ effector memory T (TEM) cells and CD11c+ antigen-presenting cells (APC) as associated with response (p = 0.0004 and 0.0255, respectively), progression-free survival (p = 0.00079 and 0.0015, respectively), and irAEs (p = 0.0034 and 0.0125, respectively) in anti-PD-1-treated patients with HCC. Type-1 conventional dendritic cells were identified as the specific APC associated with response, while 2 immunosuppressive CD14+ myeloid clusters were linked to reduced irAEs. Further analyses of CXCR3+CD8+ TEM cells showed cell-cell interactions specific to response vs. irAEs, from which the anti-PD-1 and anti-TNFR2 combination was harnessed to uncouple these effects, resulting in enhanced response without increased irAEs in a murine HCC model. CONCLUSIONS: This study identifies early predictors of clinical response to anti-PD-1 ICB in patients with HCC and offers mechanistic insights into the immune trajectories of these immune subsets at the interface between response and toxicity. We also propose a new combination immunotherapy for HCC to enhance response without exacerbating irAEs. CLINICAL TRIAL NUMBER: NCT03695952. LAY SUMMARY: Response rates to immune checkpoint blockade (ICB) treatment in hepatocellular carcinoma (HCC) remain modest and adverse events are common. Herein, we identified early predictors of response and gained an in-depth understanding of the immunological mechanisms behind response and adverse events in patients with HCC treated with ICB. We also proposed a new combination immunotherapy for HCC that enhances response without exacerbating adverse events.

Non-terminally exhausted tumor-resident memory HBV-specific T cell responses correlate with relapse-free survival in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) often develops following chronic hepatitis B virus (HBV) infection and responds poorly to immune checkpoint blockade. Here, we examined the antigen specificities of HCC-infiltrating T cells and their relevance to tumor control. Using highly multiplexed peptide-MHC tetramer staining of unexpanded cells from blood, liver, and tumor tissues from 46 HCC patients, we detected 91 different antigen-specific CD8+ T cell populations targeting HBV, neoantigen, tumor-associated, and disease-unrelated antigens. Parallel high-dimensional analysis delineated five distinct antigen-specific tissue-resident memory T (Trm) cell populations. Intratumoral and intrahepatic HBV-specific T cells were enriched for two Trm cell subsets that were PD-1loTOXlo, despite being clonally expanded. High frequencies of intratumoral terminally exhausted T cells were uncommon. Patients with tumor-infiltrating HBV-specific CD8+ Trm cells exhibited longer-term relapse-free survival. Thus, non-terminally exhausted HBV-specific CD8+ Trm cells show hallmarks of active involvement and effective antitumor response, implying that these cells could be harnessed for therapeutic purposes.

Correlating indoor and outdoor temperature and humidity in a sample of buildings in tropical climates.

The incidence of several respiratory viral infections has been shown to be related to climate. Because humans spend most of their time indoors, measures of indoor climate, rather than outdoor climate, may be better predictors of disease incidence and transmission. Therefore, understanding the relationship between indoor and outdoor climate will help illuminate their influence on the seasonality of diseases caused by respiratory viruses. Indoor-outdoor relationships between temperature and humidity have been documented in temperate regions, but little information is available for tropical regions, where seasonal patterns of respiratory viral diseases differ. We have examined indoor-outdoor correlations of temperature, relative humidity (RH), and absolute humidity (AH) over a 1-year period in each of seven tropical cities. Across all cities, the average monthly indoor temperature was 25 ± 3°C (mean ± standard deviation) with a range of 20-30°C. The average monthly indoor RH was 66 ± 9% with a range of 50-78%, and the average monthly indoor AH was 15 ± 3 g/m3 with a range of 10-23 g/m3 . Indoor AH and RH were linearly correlated with outdoor AH when the air conditioning (AC) was off, suggesting that outdoor AH may be a good proxy of indoor humidity in the absence of AC. All indoor measurements were more strongly correlated with outdoor measurements as distance from the equator increased. Such correlations were weaker during the wet season, especially when AC was in operation. These correlations will provide insight for assessing the seasonality of respiratory viral infections using outdoor climate data, which is more widely available than indoor data, even though transmission of these diseases mainly occurs indoors.

Iqgap3-Ras axis drives stem cell proliferation in the stomach corpus during homoeostasis and repair.

OBJECTIVE: Tissue stem cells are central regulators of organ homoeostasis. We looked for a protein that is exclusively expressed and functionally involved in stem cell activity in rapidly proliferating isthmus stem cells in the stomach corpus. DESIGN: We uncovered the specific expression of Iqgap3 in proliferating isthmus stem cells through immunofluorescence and in situ hybridisation. We performed lineage tracing and transcriptomic analysis of Iqgap3 +isthmus stem cells with the Iqgap3-2A-tdTomato mouse model. Depletion of Iqgap3 revealed its functional importance in maintenance and proliferation of stem cells. We further studied Iqgap3 expression and the associated gene expression changes during tissue repair after tamoxifen-induced damage. Immunohistochemistry revealed elevated expression of Iqgap3 in proliferating regions of gastric tumours from patient samples. RESULTS: Iqgap3 is a highly specific marker of proliferating isthmus stem cells during homoeostasis. Iqgap3+isthmus stem cells give rise to major cell types of the corpus unit. Iqgap3 expression is essential for the maintenance of stem potential. The Ras pathway is a critical partner of Iqgap3 in promoting strong proliferation in isthmus stem cells. The robust induction of Iqgap3 expression following tissue damage indicates an active role for Iqgap3 in tissue regeneration. CONCLUSION: IQGAP3 is a major regulator of stomach epithelial tissue homoeostasis and repair. The upregulation of IQGAP3 in gastric cancer suggests that IQGAP3 plays an important role in cancer cell proliferation.

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.

Association between meteorological variations and activities of influenza A and B across different climate zones: a multi-region modelling analysis across the globe.

OBJECTIVE: To elucidate the effects of meteorological variations on the activity of influenza A and B in 11 sites across different climate regions. METHODS: Daily numbers of laboratory-confirmed influenza A and B cases from 2011-2015 were collected from study sites where the corresponding daily mean temperature, relative humidity, wind speed and daily precipitation amount were used for boosted regression trees analysis on the marginal associations and the interaction effects. RESULTS: Cold temperature was a major determinant that favored both influenza A and B in temperate and subtropical sites. Temperature-to-influenza A, but not influenza B, exhibited a U-shape association in subtropical and tropical sites. High relative humidity was also associated with influenza activities but was less consistent with influenza B activity. Compared with relative humidity, absolute humidity had a stronger association - it was negatively associated with influenza B activity in temperate zones, but was positively associated with both influenza A and B in subtropical and tropical zones. CONCLUSION: The association between meteorological factors and with influenza activity is virus type specific and climate dependent. The heavy influence of temperature on influenza activity across climate zones implies that global warming is likely to have an impact on the influenza burden.

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.

Comparative global epidemiology of influenza, respiratory syncytial and parainfluenza viruses, 2010-2015.

OBJECTIVES: To improve our understanding of the global epidemiology of common respiratory viruses by analysing their contemporaneous incidence at multiple sites. METHODS: 2010-2015 incidence data for influenza A (IAV), influenza B (IBV), respiratory syncytial (RSV) and parainfluenza (PIV) virus infections were collected from 18 sites (14 countries), consisting of local (n = 6), regional (n = 9) and national (n = 3) laboratories using molecular diagnostic methods. Each site submitted monthly virus incidence data, together with details of their patient populations tested and diagnostic assays used. RESULTS: For the Northern Hemisphere temperate countries, the IAV, IBV and RSV incidence peaks were 2-6 months out of phase with those in the Southern Hemisphere, with IAV having a sharp out-of-phase difference at 6 months, whereas IBV and RSV showed more variable out-of-phase differences of 2-6 months. The tropical sites Singapore and Kuala Lumpur showed fluctuating incidence of these viruses throughout the year, whereas subtropical sites such as Hong Kong, Brisbane and Sydney showed distinctive biannual peaks for IAV but not for RSV and PIV. CONCLUSIONS: There was a notable pattern of synchrony of IAV, IBV and RSV incidence peaks globally, and within countries with multiple sampling sites (Canada, UK, Australia), despite significant distances between these sites.

Patterns of mutation within an emerging endemic lineage of HEV-3a.

The hepatitis E virus can cause chronic infections in immuno-suppressed patients, and cases have been on the rise globally. Viral mutations during such infections are difficult to characterize. We deep-sequenced viral populations from 15 immunocompromised patients with chronic HEV to identify the viral lineage and describe viral mutational hotspots within and across patients. A total of 21 viral RNA samples were collected between 2012 and 2017 from a single hospital in Singapore. Sequences covering a total of 3894 bp of the HEV genome were obtained. Phylogenetic analyses identified all sequences as belonging to the HEV-3a sub-clade and clearly indicate a unique local lineage. Deep sequencing reveals variable viral population complexity during infections. Comparisons of viral samples from the same patients spaced 2-19 months apart revealed rapid nucleotide replacements in the dominant viral sequence in both ribavirin treated and treatment-naive patients. Mutational hotspots were identified within ORF3 and the PCP/HVR domain of ORF1.

Evaluation of the Luminex ARIES HSV 1&2 Assay and Comparison with the FTD Neuro 9 and In-house Real-Time PCR Assays for Detecting Herpes Simplex Viruses.

BACKGROUND: Human herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) are responsible for a plethora of human diseases, of which cutaneous and mucocutaneous infections are the most prevalent. In its most severe form, HSV infection can cause meningitis/encephalitis. We compared the Luminex ARIES HSV 1&2 assay (Luminex Corp., Austin, TX, USA), an automated sample-to-result molecular solution, to two non-automated HSV DNA assays. METHODS: A total of 116 artificial controls were used to determine the analytical performance of the ARIES assay. Controls were prepared by spiking universal transport medium (UTM) and cerebrospinal fluid (CSF) samples from patients who tested negative for HSV by an in-house HSV-1 and -2 DNA assay with reference materials (SeraCare Life Sciences, MA, USA; ZeptoMetrix Corp., MA, USA). Another 117 clinical samples were then used to compare the clinical performance of the ARIES assay with those of an in-house assay and the FTD Neuro 9 assay (Fast Track Diagnostics, Junglinster, Luxembourg). RESULTS: The analytical sensitivity (95% limit of detection) of the ARIES assay was 318 copies/mL (UTM samples) and 935 copies/mL (CSF samples) for HSV-1 strain 96 and 253 copies/mL (UTM samples) and 821 copies/mL (CSF samples) for HSV-2 strain 09. No cross-reactivity was observed in samples spiked with 14 non-HSV microorganisms. Compared with the reference result (agreement between the in-house and FTD Neuro 9 results), the ARIES assay had overall concordance rates of 98.2% (111/113) and 100% (113/113) for HSV-1 and HSV-2, respectively. CONCLUSIONS: The ARIES assay appears to be an excellent alternative for rapid detection and differentiation of HSV in skin and genital infections, meningitis, and encephalitis.

Cost-benefit analysis of introducing next-generation sequencing (metagenomic) pathogen testing in the setting of pyrexia of unknown origin.

Pyrexia of unknown origin (PUO) is defined as a temperature of >38.3°C that lasts for >3 weeks, where no cause can be found despite appropriate investigation. Existing protocols for the work-up of PUO can be extensive and costly, motivating the application of recent advances in molecular diagnostics to pathogen testing. There have been many reports describing various analytical methods and performance of metagenomic pathogen testing in clinical samples but the economics of it has been less well studied. This study pragmatically evaluates the feasibility of introducing metagenomic testing in this setting by assessing the relative cost of clinically-relevant strategies employing this investigative tool under various cost and performance scenarios using Singapore as a demonstration case, and assessing the price and performance benchmarks, which would need to be achieved for metagenomic testing to be potentially considered financially viable relative to the current diagnostic standard. This study has some important limitations: we examined only impact of introducing the metagenomic test to the overall diagnostic cost and excluded costs associated with hospitalization and makes assumptions about the performance of the routine diagnostic tests, limiting the cost of metagenomic test, and the lack of further work-up after positive pathogen detection by the metagenomic test. However, these assumptions were necessary to keep the model within reasonable limits. In spite of these, the simplified presentation lends itself to the illustration of the key insights of our paper. In general, we find the use of metagenomic testing as second-line investigation is effectively dominated, and that use of metagenomic testing at first-line would typically require higher rates of detection or lower cost than currently available in order to be justifiable purely as a cost-saving measure. We conclude that current conditions do not warrant a widespread rush to deploy metagenomic testing to resolve any and all uncertainty, but rather as a front-line technology that should be used in specific contexts, as a supplement to rather than a replacement for careful clinical judgement.

Comparison of cobas HCV GT against Versant HCV Genotype 2.0 (LiPA) with confirmation by Sanger sequencing.

BACKGROUND: Correct identification of infecting hepatitis C virus (HCV) genotype is helpful for targeted antiviral therapy. OBJECTIVES: Here, we compared the HCV genotyping performance of the cobas HCV GT assay against the Versant HCV Genotype 2.0 (LiPA) assay, using 97 archived serum samples. STUDY DESIGN: In the event of discrepant or indeterminate results produced by either assay, the core and NS5B regions were sequenced. RESULTS: Of the 97 samples tested by the cobas, 25 (26%) were deemed indeterminate. Sequencing analyses confirmed 21 (84%) of the 25 samples as genotype 6 viruses with either subtype 6m, 6n, 6v, 6xa, or unknown subtype. Of the 97 samples tested by the LiPA, thirteen (13%) were deemed indeterminate. Seven (7%) were assigned with genotype 1, with unavailable/inconclusive results from the core region of the LiPA. Notably, the 7 samples were later found to be either genotype 3 or 6 by sequencing analyses. Moreover, 1 sample by the LiPA was assigned as genotypes 4 (cobas: indeterminate) but were later found to be genotype 3 by sequencing analyses, highlighting its limitation in assigning the correct genotype. CONCLUSIONS: The cobas showed similar or slightly higher accuracy (100%; 95% CI 94-100%) compared to the LiPA (99%; 95% CI 92-100%). Twenty-six percent of the 97 samples tested by the cobas had indeterminate results, mainly due to its limitation in identifying genotype 6 other than subtypes 6a and 6b. This presents a significant assay limitation in Southeast Asia, where genotype 6 infection is highly prevalent.

CEBPA mutational analysis in acute myeloid leukaemia by a laboratory-developed next-generation sequencing assay.

AIM: The presence of biallelic CEBPA mutations is a favourable prognostic feature in acute myeloid leukaemia (AML). CEBPA mutations are currently identified through conventional capillary sequencing (CCS). With the increasing adoption of next-generation sequencing (NGS) platforms, challenges with regard to amplification efficiency of CEBPA due to the high GC content may be encountered, potentially resulting in suboptimal coverage. Here, the performance of an amplicon-based NGS method using a laboratory-developed CEBPA-specific Nextera XT (CEBNX) was evaluated. METHODS: Mutational analyses of the CEBPA gene of 137 AML bone marrow or peripheral blood retrospective specimens were performed by the amplification of the CEBPA gene using the Expand Long Range dNTPack and the amplicons processed by CCS and NGS. CEBPA-specific libraries were then constructed using the Nextera XT V.2 kit. All FASTQ files were then processed with the MiSeq Reporter V.2.6.2.3 using the PCR Amplicon workflow via the customised CEBPA-specific manifest file. The variant calling format files were analysed using the Illumina Variant Studio V.2.2. RESULTS: A coverage per base of 3631X to 28184X was achieved. 22 samples (16.1%) were found to contain CEBPA mutations, with variant allele frequencies (VAF) ranging from 3.8% to 58.2%. Taking CCS as the 'gold standard', sensitivity and specificity of 97% and 97% was achieved. For the transactivation domain 2 polymorphism (c.584_589dupACCCGC/p.His195_Pro196dup), the CEBNX achieved 100% sensitivity and 100% specificity relative to CCS. CONCLUSIONS: Our laboratory-developed CEBNX workflow shows high coverage and thus overcomes the challenges associated with amplification efficiency and low coverage of CEBPA. Therefore, our assay is suitable for deployment in the clinical laboratory.

Performance evaluation of Cepheid Xpert Norovirus kit with a user-modified protocol.

The Cepheid Xpert® Norovirus kit automates sample processing, nucleic acid extraction, and real-time reverse transcription polymerase chain reactions (RT-PCRs) to detect norovirus genogroups I (GI) and II (GII). Eighty-five stool samples collected between February 2015 and May 2017 were used to compare the performance of a user-modified Xpert assay against a clinically validated laboratory-developed test (LDT). Of the 85 samples, 54 were previously archived in -80°C freezer. The remaining 31 were fresh samples tested concurrently with the LDT. The results of all samples tested using the Xpert kit and LDT were found to be concordant, including 12 GI- and 42 GII-positive samples, 1 GI and GII coinfection, and 30 negative samples. Comparison of the assays showed perfect concordance with a kappa coefficient score of 1.00 (95%CI from 1.00 to 1.00). Of the 30 negative stool samples tested, three samples were positive for rotavirus detected using an immunochromatographic assay, with no cross-reactivity shown in both LDT and Xpert assays. In-run sample processing control of the Xpert assay for all negative samples tested showed no/minor inhibition. Compared to the LDT, the Xpert assay produced similar or better Ct values for detection. It also showed better mitigation of PCR inhibition in stool sample testing.

Comparison of Luminex NxTAG Respiratory Pathogen Panel and xTAG Respiratory Viral Panel FAST Version 2 for the Detection of Respiratory Viruses.

Owing to advancements in molecular diagnostics, recent years have seen an increasing number of laboratories adopting respiratory viral panels to detect respiratory pathogens. In December 2015, the NxTAG respiratory pathogen panel (NxTAG RPP) was approved by the United States Food and Drug Administration. We compared the clinical performance of this new assay with that of the xTAG respiratory viral panel (xTAG RVP) FAST v2 using 142 clinical samples and 12 external quality assessment samples. Discordant results were resolved by using a laboratory-developed respiratory viral panel. The NxTAG RPP achieved 100% concordant negative results and 86.6% concordant positive results. It detected one coronavirus 229E and eight influenza A/H3N2 viruses that were missed by the xTAG RVP FAST v2. On the other hand, the NxTAG RPP missed one enterovirus/rhinovirus and one metapneumovirus that were detected by FAST v2. Both panels correctly identified all the pathogens in the 12 external quality assessment samples. Overall, the NxTAG RPP demonstrated good diagnostic performance. Of note, it was better able to subtype the influenza A/H3N2 viruses compared with the xTAG RVP FAST v2.

Molecular Evidence of Transmission of Influenza A/H1N1 2009 on a University Campus.

BACKGROUND: In the recent years, the data on the molecular epidemiology of influenza viruses have expanded enormously because of the availability of cutting-edge sequencing technologies. However, much of the information is from the temperate regions with few studies from tropical regions such as South-east Asia. Despite the fact that influenza has been known to transmit rapidly within semi-closed communities, such as military camps and educational institutions, data are limited from these communities. OBJECTIVES: To determine the phylogeography of influenza viruses on a university campus, we examined the spatial distribution of influenza virus on the National University of Singapore (NUS) campus. METHODS: Consenting students from the NUS who sought medical attention at the UHC provided two nasopharyngeal swabs and demographic data. PCR was used for detection of influenza viruses. 34 full-genomes of pH1N1/09 viruses were successfully sequenced by Sanger method and concatenated using Geneious R7. Phylogenetic analysis was conducted using these 34 sequences and 1518 global sequences. Phylogeographic analysis was done using BaTS software and Association index and Fitch parsimony scores were determined. RESULTS: Integrating whole genome sequencing data with epidemiological data, we found strong evidence of influenza transmission on campus as isolates from students residing on-campus were highly similar to each other (AI, P value = 0.009; PS, P value = 0.04). There was also evidence of multiple introductions from the community. CONCLUSIONS: Such data are useful in formulating pandemic preparedness plans which can use these communities as sentinel sites for detection and monitoring of emerging respiratory viral infections.

Contamination-controlled high-throughput whole genome sequencing for influenza A viruses using the MiSeq sequencer.

Accurate full-length genomic sequences are important for viral phylogenetic studies. We developed a targeted high-throughput whole genome sequencing (HT-WGS) method for influenza A viruses, which utilized an enzymatic cleavage-based approach, the Nextera XT DNA library preparation kit, for library preparation. The entire library preparation workflow was adapted for the Sentosa SX101, a liquid handling platform, to automate this labor-intensive step. As the enzymatic cleavage-based approach generates low coverage reads at both ends of the cleaved products, we corrected this loss of sequencing coverage at the termini by introducing modified primers during the targeted amplification step to generate full-length influenza A sequences with even coverage across the whole genome. Another challenge of targeted HTS is the risk of specimen-to-specimen cross-contamination during the library preparation step that results in the calling of false-positive minority variants. We included an in-run, negative system control to capture contamination reads that may be generated during the liquid handling procedures. The upper limits of 99.99% prediction intervals of the contamination rate were adopted as cut-off values of contamination reads. Here, 148 influenza A/H3N2 samples were sequenced using the HTS protocol and were compared against a Sanger-based sequencing method. Our data showed that the rate of specimen-to-specimen cross-contamination was highly significant in HTS.