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.

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.

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.

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.

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.

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.

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.

The Use of Polymerase Chain Reaction Amplification for the Detection of Viruses and Bacteria in Severe Community-Acquired Pneumonia.

BACKGROUND: Pathogens are often not identified in severe community-acquired pneumonia (CAP), and the few studies using polymerase chain reaction (PCR) techniques for virus detection are from temperate countries. OBJECTIVE: This study assesses if PCR amplification improves virus and bacteria detection, and if viral infection contributes to mortality in severe CAP in a tropical setting, where respiratory pathogens have less well-defined seasonality. METHODS: In this cohort study of patients with severe CAP in an intensive care unit, endotracheal aspirates for intubated patients and nasopharyngeal swabs for non-intubated patients were sent for PCR amplification for respiratory viruses. Blood, endotracheal aspirates for intubated patients, and sputum for non-intubated patients were analysed using a multiplex PCR system for bacteria. RESULTS: Out of 100 patients, using predominantly cultures, bacteria were identified in 42 patients; PCR amplification increased this number to 55 patients. PCR amplification identified viruses in 32 patients. In total, only bacteria, only viruses, and both bacteria and viruses were found in 37, 14, and 18 patients, respectively. The commonest viruses were influenza A H1N1/2009 and rhinovirus; the commonest bacterium was Streptococcus pneumoniae. Hospital mortality rates for patients with no pathogens, bacterial infection, viral infection, and bacterial-viral co-infection were 16.1, 24.3, 0, and 5.6%, respectively (p = 0.10). On multivariable analysis, virus detection was associated with lower mortality (adjusted odds ratio 0.12, 95% confidence interval 0.2-0.99; p = 0.049). CONCLUSIONS: Viruses and bacteria were detected in 7 of 10 patients with severe CAP with the aid of PCR amplification. Viral infection appears to be independently associated with lower mortality.

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.

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/ ).

An in-house HIV genotyping assay for the detection of drug resistance mutations in Southeast Asian patients infected with HIV-1.

Genotyping for HIV drug resistance is costly and beyond the means for many Southeast Asian patients, who are self-funded. This prompted the development of a more cost-effective, in-house assay for an ethnically diverse, Southeast Asian population at the National University Hospital in Singapore, using Sanger-based sequencing. Plasma samples from 20 treatment-failure patients with a broad spectrum of HIV drug resistance mutations were used to validate this assay clinically. Blinded testing gave concordant results for 7/7 (100%) protease drug resistance-related mutations, including one major and six minor mutations, and 111/116 (95.7%) reverse-transcriptase (RT) drug resistance-related mutations, including 65 nucleoside RT inhibitors (NRTI) and 46 non-nucleoside RT inhibitors (NNRTI) mutations. There were five discordant results, involving three NRTI- and two NNRTI-resistance-associated mutations. Highly conserved primers designed to have a wide coverage of the HIV pol gene (covering the entire protease and 395 codons of the RT region) enabled efficient multi-ethnic population-based genotyping. Reagents for this in-house test cost around 60% less than those for commercially available assays (SGD150 vs. SGD350 per sample). In addition, this assay also identified mutations located within the C-terminal domain (codons 312-560) of RT that are beyond the reach of most published and commercial GRTs. Currently, most research on C-terminal drug-resistance-related mutations has been conducted on HIV subtype B infections. Therefore this assay enables further study of these C-terminal mutations in Southeast Asian populations, where there is a high prevalence of CRF01_AE and other non-subtype B HIV infections.

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 C(q) values correlated well between the pre- and post-revision protocols for influenza A (r(2) = 0.998) and B (r(2) = 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.

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.

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.

High-resolution melting approach to efficient identification and quantification of H275Y mutant influenza H1N1/2009 virus in mixed-virus-population samples.

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 (C(T)) 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 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.

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.

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.

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.