Opt-out HIV screening in adults attending the Emergency Department of a London teaching hospital.

BACKGROUND: Opt-out Emergency Department blood borne virus (EDBBV) screening was introduced at the Royal Free Hospital under the NHSEI (NHS England and NHS Improvement) programme to expand opt-out testing in local authority areas with high HIV prevalence. This initiative was part of the "Toward Zero" policy towards ending HIV transmission in England by 2030. METHODS: All patients attending the Royal Free Hospital Emergency Department (ED) aged 16 and over were screened for blood borne viruses (HIV/HBV/HCV) unless they opted out. We looked at HIV data from patients seen in ED between the initiation of EDBBV testing on the 12th of April and 12th of August 2022. Hepatitis B and C data was reviewed in a separate study. OUTCOME: A total of 12,208 samples from 10,641 patients were screened for HIV. Amongst these samples there were 88 which were positive, giving a seroprevalence of 0.84 %. There were 48 patients who were already known to local HIV services, 35 were known to HIV services outside of our Trust and 5 were new diagnoses. CONCLUSION: Our results confirmed our local HIV prevalence to be very high, as per the UK Health Security Agency and supports the need for HIV testing. Opt-out ED BBV screening has been a highly effective method for identifying people living with HIV who are unaware of their status.

Opt-out testing for hepatitis B and C infections in adults attending the emergency department of a large London teaching hospital.

BACKGROUND: The National Health Service (NHS) in England commissioned opt-out testing in London Emergency Departments (ED) in April 2022 to allow early identification and management of hepatitis B (HBV) and hepatitis C virus (HCV) infection in patients unaware of their infection status. METHODS: All adults over the age of 16 undergoing blood tests in the ED at the Royal Free Hospital were tested for HBV surface antigen and anti-HCV IgG unless they opted out. Data was collected between the 12th of April and 22nd of August 2022. OUTCOME: Of 11,215 patients tested for HCV, 164 patients were found to be anti-HCV IgG positive, giving a seroprevalence rate of 1.46 %. 52 of the anti-HCV IgG positive patients did not have any previous HCV serology result. 23 of the anti-HCV IgG positive patients were also HCV RNA positive giving an RNA seroprevalence of 0.21 %, and 17 of those were new diagnoses of HCV viraemia. For HBV testing, 82 (0.73 %) out of 11,192 patients tested were found to be HBsAg positive, including one patient who presented acutely with a positive HBV core IgM. 39 of the HBsAg positive patients were previously unknown to us; of these, 9 had an HBV viral load of more than 2000 IU/mL, including 3 patients with positive HBV e antigen and one patient with hepatitis D virus co-infection. CONCLUSION: Opt-out screening of HBV and HCV in ED is effective at identifying patients with previously undiagnosed viral hepatitis infection and providing an opportunity to engage them in specialist care.

Diagnostic accuracy of Abbott Architect Assay as a screening tool for human T-cell leukaemia virus type-1 and type-2 infection in a London teaching hospital with a large solid organ transplant centre.

AIM: In the United Kingdom, organ donors/recipients are screened for evidence of human T-cell leukaemia virus type-1 and type-2 (HTLV-1/2) infections. Since the United Kingdom is a low prevalence country for HTLV infections, a screening assay with high sensitivity and specificity is required. Samples with repeat reactivity on antibody testing are sent to a reference lab for confirmatory serological and molecular testing. In the case of donor screen, this leads to delays in the release of organs and can result in wastage. We aim to assess whether a signal/cut-off (S/CO) ratio higher than the manufacturer's recommendation of 1.0 in the Abbott Architect antibody assay is a reliable measure of HTLV-1/2 infection. METHODS: We conducted a 5 year retrospective analysis of 7245 patients from which 11 766 samples were tested on the Abbott Architect rHTLV I/II assay. Reactive samples (S/CO >1) were referred for confirmatory serological and molecular detection (Western Blot and proviral DNA) at UK Health Security Agency, (formerly PHE, Colindale), the national reference laboratory. Electronic, protected laboratory and hospital patient databases were employed to collate data. RESULTS: A total of 45 patients had initially reactive samples. 42.2% (n = 19/45) had an S/CO ratio > 20, with HTLV infection confirmed in n = 18/19 and indeterminate confirmatory results in n = 1/19. No samples with an S/CO ratio <4 (48.9%, n = 22/45) or 4-20 (8.9%, n = 4/45) had positive confirmatory results on subsequent confirmatory testing. CONCLUSION: Samples with an S/CO >20 likely represent a true HTLV-1/2 infection. Reactive samples with an S/CO <4 were unlikely to confirm for HTLV infections. Interpretation of these ratios can assist clinicians in the assessment of low reactive samples and reiterates the need for faster access to confirmatory testing.

Post-Vaccination Coronavirus Disease 2019: A Case-Control Study and Genomic Analysis of 119 Breakthrough Infections in Partially Vaccinated Individuals.

BACKGROUND: Post-vaccination infections challenge the control of the coronavirus disease 2019 (COVID-19) pandemic. METHODS: We matched 119 cases of post-vaccination severe acute respiratory syndrome coronavirus 2 infection with BNT162b2 mRNA or ChAdOx1 nCOV-19 to 476 unvaccinated patients with COVID-19 (September 2020-March 2021) according to age and sex. Differences in 60-day all-cause mortality, hospital admission, and hospital length of stay were evaluated. Phylogenetic, single-nucleotide polymorphism (SNP), and minority variant allele (MVA) full-genome sequencing analysis was performed. RESULTS: Overall, 116 of 119 cases developed COVID-19 post-first vaccination dose (median, 14 days). Thirteen of 119 (10.9%) cases and 158 of 476 (33.2%) controls died (P < .001), corresponding to the 4.5 number needed to treat (NNT). Multivariably, vaccination was associated with a 69.3% (95% confidence interval [CI]: 45.8 to 82.6) relative risk (RR) reduction in mortality. Similar results were seen in subgroup analysis for patients with infection onset ≥14 days after first vaccination and across vaccine subgroups. Hospital admissions (odds ratio, 0.80; 95% CI: .51 to 1.28) and length of stay (-1.89 days; 95% CI: -4.57 to 0.78) were lower for cases, while cycle threshold values were higher (30.8 vs 28.8, P = .053). B.1.1.7 was the predominant lineage in cases (100 of 108, 92.6%) and controls (341 of 446, 76.5%). Genomic analysis identified 1 post-vaccination case that harbored the E484K vaccine-escape mutation (B.1.525 lineage). CONCLUSIONS: Previous vaccination reduces mortality when B.1.1.7 is the predominant lineage. No significant lineage-specific genomic changes during phylogenetic, SNP, and MVA analysis were detected.

Whole genome sequencing of Herpes Simplex Virus 1 directly from human cerebrospinal fluid reveals selective constraints in neurotropic viruses.

Herpes Simplex Virus type 1 (HSV-1) chronically infects over 70 per cent of the global population. Clinical manifestations are largely restricted to recurrent epidermal vesicles. However, HSV-1 also leads to encephalitis, the infection of the brain parenchyma, with high associated rates of mortality and morbidity. In this study, we performed target enrichment followed by direct sequencing of HSV-1 genomes, using target enrichment methods on the cerebrospinal fluid (CSF) of clinical encephalitis patients and from skin swabs of epidermal vesicles on non-encephalopathic patients. Phylogenetic analysis revealed high inter-host diversity and little population structure. In contrast, samples from different lesions in the same patient clustered with similar patterns of allelic variants. Comparison of consensus genome sequences shows HSV-1 has been freely recombining, except for distinct islands of linkage disequilibrium (LD). This suggests functional constraints prevent recombination between certain genes, notably those encoding pairs of interacting proteins. Distinct LD patterns characterised subsets of viruses recovered from CSF and skin lesions, which may reflect different evolutionary constraints in different body compartments. Functions of genes under differential constraint related to immunity or tropism and provide new hypotheses on tissue-specific mechanisms of viral infection and latency.

Cytomegalovirus viral load parameters associated with earlier initiation of pre-emptive therapy after solid organ transplantation.

BACKGROUND: Human cytomegalovirus (HCMV) can be managed by monitoring HCMV DNA in the blood and giving valganciclovir when viral load exceeds a defined value. We hypothesised that such pre-emptive therapy should occur earlier than the standard 3000 genomes/ml (2520 IU/ml) when a seropositive donor transmitted virus to a seronegative recipient (D+R-) following solid organ transplantation (SOT). METHODS: Our local protocol was changed so that D+R- SOT patients commenced valganciclovir once the viral load exceeded 200 genomes/ml; 168 IU/ml (new protocol). The decision point remained at 3000 genomes/ml (old protocol) for the other two patient subgroups (D+R+, D-R+). Virological outcomes were assessed three years later, when 74 D+R- patients treated under the old protocol could be compared with 67 treated afterwards. The primary outcomes were changes in peak viral load, duration of viraemia and duration of treatment in the D+R- group. The secondary outcome was the proportion of D+R- patients who developed subsequent viraemia episodes. FINDINGS: In the D+R- patients, the median values of peak viral load (30,774 to 11,135 genomes/ml, p<0.0215) were significantly reduced on the new protocol compared to the old, but the duration of viraemia and duration of treatment were not. Early treatment increased subsequent episodes of viraemia from 33/58 (57%) to 36/49 (73%) of patients (p< 0.0743) with a significant increase (p = 0.0072) in those episodes that required treatment (16/58; 27% versus 26/49; 53%). Median peak viral load increased significantly (2,103 to 3,934 genomes/ml, p<0.0249) in the D+R+ but not in the D-R+ patient subgroups. There was no change in duration of viraemia or duration of treatment for any patient subgroup. INTERPRETATION: Pre-emptive therapy initiated at the first sign of viraemia post-transplant significantly reduced the peak viral load but increased later episodes of viraemia, consistent with the hypothesis of reduced antigenic stimulation of the immune system.

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.

Respiratory Infections and Antibiotic Usage in Common Variable Immunodeficiency.

BACKGROUND: Patients with common variable immunodeficiency (CVID) suffer frequent respiratory tract infections despite immunoglobulin replacement and are prescribed significant quantities of antibiotics. The clinical and microbiological nature of these exacerbations, the symptomatic triggers to take antibiotics, and the response to treatment have not been previously investigated. OBJECTIVES: To describe the nature, frequency, treatment, and clinical course of respiratory tract exacerbations in patients with CVID and to describe pathogens isolated during respiratory tract exacerbations. METHODS: We performed a prospective diary card exercise in 69 patients with CVID recruited from a primary immunodeficiency clinic in the United Kingdom, generating 6210 days of symptom data. We collected microbiology (sputum microscopy and culture, atypical bacterial PCR, and mycobacterial culture) and virology (nasopharyngeal swab multiplex PCR) samples from symptomatic patients with CVID. RESULTS: There were 170 symptomatic exacerbations and 76 exacerbations treated by antibiotics. The strongest symptomatic predictors for commencing antibiotics were cough, shortness of breath, and purulent sputum. There was a median delay of 5 days from the onset of symptoms to commencing antibiotics. Episodes characterized by purulent sputum responded more quickly to antibiotics, whereas sore throat and upper respiratory tract symptoms responded less quickly. A pathogenic virus was isolated in 56% of respiratory exacerbations and a potentially pathogenic bacteria in 33%. CONCLUSIONS: Patients with CVID delay and avoid treatment of symptomatic respiratory exacerbations, which could result in structural lung damage. However, viruses are commonly represented and illnesses dominated by upper respiratory tract symptoms respond poorly to antibiotics, suggesting that antibiotic usage could be better targeted.

A deletion at CMV UL54 codon 524 without co-existing resistance-associated mutation at UL97 confers resistance to ganciclovir: A case report.

Sequencing of Cytomegalovirus (CMV) genes to investigate antiviral resistance is a growing area of interest as treatment for CMV infection becomes more widely available and used. Using conventional sequencing methods, we identified a deletion in UL54 gene, del524, which conferred resistance to ganciclovir in a renal transplant recipient, in the absence of a co-existing resistance-conferring mutation in UL97 gene. This case report reinforces that both UL97 and UL54 genes should be sequenced when exploring CMV antiviral resistance as mutations have been identified in both genes independently of each other.

Transmission of Hepatitis B Core Antibody and Galactomannan Enzyme Immunoassay Positivity via Immunoglobulin Products: A Comprehensive Analysis.

BACKGROUND: Therapeutic immunoglobulins are used as replacement or immunomodulatory therapy, but can transmit clinically important molecules. We investigated hepatitis B virus (HBV) antibodies and galactomannan enzyme immunoassay (GM-EIA) positivity. Detection of HBV core antibody may prompt antiviral prophylaxis when commencing therapy such as rituximab; a positive GM-EIA result prompts investigation or treatment for invasive fungal disease. METHODS: We performed a cross-sectional analysis of HBV serology in 80 patients established (>6 months) on immunoglobulin therapy; prospective analysis of HBV serology in 16 patients commencing intravenous immunoglobulin (IVIG); and pre- and post-infusion analysis of GM-EIA in 37 patients receiving IVIG. RESULTS: Pre-IVIG, 9 of 80 patients tested positive for HBV surface antibody and 1 of 80 tested equivocal for HBV core antibody. On IVIG, 79 of 79 tested positive for surface antibody, 37 of 80 tested positive for core antibody, and 10 of 80 tested equivocal for core antibody. There were significant differences by product, but among patients receiving products that appear to transmit core antibody, negative results correlated with lower surface antibody titers and longer time since infusion, suggesting a simple concentration effect. There was a progressive increase with each infusion in the percentage of patients testing positive for HBV core antibody among patients newly commencing IVIG. Some patients "seroreverted" to negative during therapy. Certain IVIG products tested positive for GM-EIA and there were rises in index values in corresponding patient samples from pre- to post-infusion. Overall, 5 of 37 patient samples pre-infusion and 15 of 37 samples post-infusion tested positive for GM-EIA. CONCLUSIONS: HBV antibodies and GM-EIA positivity are common in patients receiving IVIG and confound diagnostic results.

HIV co-receptor tropism prediction remains stable over time in treatment-naïve patients.

HIV co-receptor tropism determination is essential before prescribing the CCR5 antagonist maraviroc. British HIV Association guidelines suggest tropism testing may remain valid for only 90 days in antiretroviral-naïve patients. We aimed to determine the accuracy of this figure. Tropism was assessed in 26 antiretroviral-naïve patients with ongoing viral replication, sampled yearly from first clinic visit. The V3 region of HIV-1 was sequenced in triplicate, then tropism predicted using the Geno2Pheno system. Baseline tropism prediction remained valid for a median of 52 months (range 7-81). For 19/26 individuals baseline tropism remained unchanged throughout a median of 54 months follow-up; 18 R5 tropic and 1 X4 tropic. In seven patients (27%) baseline tropism switched at least once (range 1-4 switches) during follow-up; however, their baseline tropism prediction remained valid for a median of 45 months. Co-receptor tropism in treatment-naïve patients with ongoing viral replication appears highly stable over time, suggesting that baseline genotypic tropism prediction may be valid for a longer duration in patients delaying ART initiation. In this study, baseline tropism prediction remained valid for a median of 52 months, suggesting current guidelines recommending repeat testing after 90 days may be excessively conservative in their assessment of tropism stability.

IL28B genotype predicts response to chronic hepatitis C triple therapy with telaprevir or boceprevir in treatment naïve and treatment-experienced patients other than prior partial- and null-responders.

Single nucleotide polymorphisms (SNPs) in the IL28B gene were shown to have limited utility in predicting response to telaprevir and boceprevir in treatment of chronic HCV infection in clinical trials. Data outside of the clinical trial setting are lacking. We assessed the value of single and combined IL28B SNPs rs12979860 and rs8099917 genotypes in predicting sustained virological response 12 weeks after cessation of triple therapy (SVR12) with telaprevir or boceprevir in a single-centre cohort of treatment-naïve and treatment-experienced patients with genotype 1 HCV mono-infection (n = 105). The overall SVR12 rate was 65.7%. By unadjusted bivariate logistic regression analysis, rs12979860-CC and rs8099917-TT were significantly associated with SVR12 in the subgroup of patients including all naïve patients and all treatment-experienced patients with the exception of partial- and null-responders to previous HCV therapy. The predictive value of rs12979860-CC was stronger than rs8099917-TT and only rs12979860-CC remained significantly predictive of treatment success when the two variants were assessed by adjusted logistic regression analysis in the whole study cohort. In patients presenting the rs12979860-CC variant, the additional determination of rs8099917 genotype had no value. IL28B rs12979860-CC remained significantly associated with SVR12 also in the multivariate analysis including the other baseline characteristics associated to SVR12 in the bivariate analysis (i.e., female gender, HCV genotype 1b, baseline viral load <800,000 IU/mL, advanced liver fibrosis and prior partial- or null-response to HCV therapy). Our study suggests that testing for the IL28B rs12979860 genotype may still be useful in predicting response to triple therapy with boceprevir and telaprevir in naïve patients and treatment-experienced patients other than partial and null-responders.

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.

Evaluation of sequencing of HCV core/E1, NS5A and NS5B as a genotype predictive tool in comparison with commercial assays targeting 5'UTR.

BACKGROUND: Hepatitis C virus (HCV) genotyping is required for tailoring the dose and duration of antiviral therapy, predicting virological response rates, and selecting future treatment options. OBJECTIVE: To establish whether baseline genotypes, performed by INNO-LiPA Version 1.0 (v1.0), before 2008, were valid for making treatment decisions now or whether genotypic determination should be repeated. Furthermore, to evaluate concordance between Abbott RealTime genotype II assay (RT) and genotyping by sequencing HCV C/E1, NS5A, NS5B. STUDY DESIGN: Genotyping by RT and sequencing was performed on paired historic and current specimens from 50 patients previously baseline genotyped using INNO-LiPA. RESULTS: Of 100 samples from 50 patients, ≥ 2 of HCV genomic target regions yielded a sequence that was suitable for genotyping, with 100% concordance, providing no evidence of recombination events. Genotype and subtype prediction based on RT and sequencing agreed in 62.8% historic and 72.7% current specimens, with a kappa coefficient score of 0.48 and 0.76, respectively. LiPA could not subtype 46% of HCV gt1 infections, and LiPA subgenotype was only in agreement with RT and sequencing in 28.6% cases, where matched baseline and historic specimens were available. Three patients were indeterminate by RT, and five patients with HCV gt1 infections could not be subtyped by RT. However, RT revealed mixed infections in five patients where sequencing detected only single HCV infection at 20% threshold. CONCLUSION: Genotyping by sequencing, exhibited excellent concordance, with moderate to good agreement with RT, and could resolve RT indeterminates and subtype HCV-gt1 infections not possible by LiPA.

The prevalence of occult hepatitis B virus (HBV) infection in a large multi-ethnic haemodialysis cohort.

BACKGROUND: Haemodialysis patients are at increased risk of exposure to blood borne viruses. To reduce transmission in the UK, all haemodialysis patients are regularly screened, and if susceptible to Hepatitis B virus (HBV) infection, vaccinated. METHODS: This retrospective study was undertaken to determine the HBV immune status in a large dialysis cohort and the prevalence of occult HBV infection, defined as the presence of anti-HBcore antibody (anti-HBcAb) and HBV DNA without detectable HB surface antigen (HBsAg). Information on HBV status was retrieved from haemodialysis patients under the care of The Royal Free Hospital, London, UK between 2009-2010. Available sera from 138 of 161 anti-HBcAb positive/HBsAg negative individuals were anonymised and tested for HBV DNA by a real time quantitative PCR. RESULTS: 15 (2%) of 793 patients had chronic HBV infection (HBsAg positive). 161 (20%) were anti-HBcAb positive but HBsAg negative suggesting past infection. 335 (54%) of the remaining 617 patients were considered immune following vaccination (anti-HBsAb > 10 IU/L). Three (2.2%) of the 138 anti-HBcAb positive, HBsAg negative patients had detectable HBV DNA (3, 5 and 9 IU/ml). Standard liver function tests were normal in these patients. CONCLUSIONS: In a large multi-ethnic London haemodialysis cohort, 20% patients had evidence of past HBV infection. Despite this, the prevalence of occult HBV was found to be low and the very low levels of HBV DNA detected are unlikely to pose a nosocomial transmission risk in the presence of robust vaccination and infection control measures.

Prevalence of baseline polymorphisms for potential resistance to NS5A inhibitors in drug-naive individuals infected with hepatitis C genotypes 1-4.

BACKGROUND: The non-structural 5A (NS5A) protein of HCV is a multifunctional phosphoprotein involved in regulation of viral replication and virion assembly. NS5A inhibitors targeting domain I of NS5A protein have demonstrated high potency and pan-genotypic antiviral activity, however they possess a low genetic barrier to resistance. At present, only genotype 1, the most prevalent HCV genotype has been studied in detail for resistant variants. METHODS: Utilizing a panel of genotypic-specific resistance assays, population sequencing was performed on plasma-derived viral RNA isolated from 138 patients infected with HCV genotypes 1-4 and not treated with direct-acting antiviral agents. Amino acid changes in HCV NS5A domain I at codon positions 28, 30, 31, 32 and 93, reported to confer reduced susceptibility to certain NS5A inhibitors were examined. Additionally, genotypic outcome based on NS5A sequences were compared with VERSANT HCV Genotype Assay (LiPA) 1.0 (Siemens Healthcare Diagnostics, Surrey, UK) and Abbott m2000 RealTime HCV genotype II assay (Abbott Molecular, Maidenhead, Berkshire, UK). RESULTS: Amino acid substitutions associated with moderate to high level resistance to NS5A inhibitors were detected in 2/42 (4.76%) HCV-1a, 3/23 (13.04%) HCV-1b, 4/26 (15.38%) HCV-2, 1/24 (4.17%) HCV-3 and 1/23 (4.35%) HCV-4 infected patients who had not been treated with NS5A inhibitors. Genotype prediction based on NS5A sequences were concordant with LiPA and/or Abbott RealTime for 97.10% of cases. CONCLUSIONS: Primary resistance mutations associated with resistance to first-generation NS5A inhibitors such as daclatasvir were observed in all genotypes, albeit at low frequencies. An excellent correlation based on NS5A genotyping and LiPA or Abbott RealTime was achieved.

Analysis of transmitted HIV-1 drug resistance using 454 ultra-deep-sequencing and the DeepChek(®)-HIV system.

INTRODUCTION: Next-generation sequencing (NGS) is capable of detecting resistance-associated mutations (RAMs) present at frequencies of 1% or below. Several studies have found that baseline low-frequency RAMs are associated with failure to first-line HAART. One major limitation to the expansion of this technology in routine diagnostics is the complexity and laboriousness integral to bioinformatics analysis. DeepChek (ABL, TherapyEdge) is a CE-marked software that allows automated analysis and resistance interpretation of NGS data. OBJECTIVE: To evaluate the use of 454 ultra-deep-sequencing (Roche(®) 454, Life Sciences; 454-UDS) and DeepChek for routine baseline resistance testing in a clinical diagnostic laboratory. METHODS: 107 newly diagnosed HIV-1-infected patients (subtypes: A, n=9; B, n=52; C, n=21; D, n=2; F, n=3; G, n=1; CRF01, n=7; CRF02, n=7; CRF06, n=1; CRF07, n=1; CRF10, n=1 and unassigned complex, n=2) with a median plasma viral load of 88,727 copies/mL (range: 1380-2,143,543) were tested by 454-UDS and Sanger sequencing for the detection of protease and reverse transcriptase RAMs. In addition, integrase RAMs were investigated in 57 of them. Sequence analysis and resistance interpretation were performed using DeepChek applying 1% and 20% thresholds for variant detections; filters applied were comparison between Sanger and 454-UDS, and Stanford and IAS list for resistance interpretation. RESULTS: The time elapsed from generation of raw 454 data (between 2,000-5,000 sequences/sample) to elaboration of a resistance report was approximately 10 minutes per sample, equivalent to the time required for the same process using Sanger sequencing. Four patients (3.7%) showed baseline resistance by Sanger and 454-UDS at frequencies above 20%, which affected both NRTIs (n=2) and NNRTIs (n=2). In addition, 12 patients (11.2%) showed transmitted drug resistance (TDR) by 454-UDS at frequencies below 20% affecting NRTIs (n=9), NNRTIs (n=7) and PIs (n=2). Integrase resistance was not detected at baseline by 454-UDS or Sanger sequencing. CONCLUSIONS: DeepChek allowed easy and rapid analysis and interpretation of NGS data, thus facilitating the incorporation of this technology in routine diagnostics. The use of NGS considerably increased the detection rates of TDR to NRTI, NNRTIs and PIs. No transmitted resistance to integrase inhibitors was found in our population by Sanger sequencing or UDS.