Evaluation of two RT-PCR screening assays for identifying SARS-CoV-2 variants.

BACKGROUND: The recent emergence of new SARS CoV-2 variants (variants of concern, VOC) that spread rapidly and may lead to immune escape has emphasized the urgent need to monitor and control their spread. METHODS: We analyzed 2018 SARS-CoV-2 positive specimens collected between February 9 and March 22, 2021 using the Thermofisher® TaqPath™ COVID-19 CE-IVD RT-PCR kit (TaqPath) and the ID solutions® ID™ SARS-CoV-2/UK/SA Variant Triplex RT-PCR (ID triplex) assay to screen for VOCs. RESULTS: The ID triplex assay identified 62.8% of them as VOCs: 61.8% B.1.1.7 and 0.9% B.1.351/P.1. The agreement between the ID triplex results for B.1.1.7 and the TaqPath S gene target failure (SGTF)/ S gene target late detection (SGTL) profile for this variant agreed very well (k = 0.86). A low virus load was the main cause of discrepancies. Sequencing discordant results with both assays indicated that the TaqPath assay detected the B.1.1.7 lineage slightly better. Both assays suggested that the virus loads of B.1.1.7 variants were significantly higher than those of non-B.1.1.7 strains. Only 10/20 B1.351/P.1 strains detected with the ID triplex assay were confirmed by sequencing. CONCLUSIONS: We conclude that the SGTF/SGTL profiles identified using the TaqPath assay and ID triplex results are suitable for detecting the B.1.1.7 lineage. The ID triplex assay, which rapidly determines all three current VOCs simultaneously, could be a valuable tool for limiting virus spread by supporting contact-tracing and isolation.

A Model-Based Illustrative Exploratory Approach to Optimize the Dosing of Peg-IFN/RBV in Cirrhotic Hepatitis C Patients Treated With Triple Therapy.

Hézode et al. recently reported the frequent occurrence of anemia and thrombocytopenia in the ANRS-CO20-CUPIC cohort of hepatitis C virus (HCV) cirrhotic experienced patients treated with pegylated-interferon (Peg-IFN), ribavirin (RBV), and telaprevir or boceprevir.1,2 Using frequent measurements of serum drug concentrations, hemoglobin, and platelet concentrations obtained in 15 patients of this cohort, we show how an on-treatment model-based approach could be used to individualize dose regimen and avoid the occurrence of RBV-induced anemia and Peg-IFN-induced thrombocytopenia.

Minority resistant HIV-1 variants and the response to first-line NNRTI therapy.

BACKGROUND: The presence of low-frequency HIV-1 variants with mutations making them resistant to non-nucleoside reverse-transcriptase inhibitors (NNRTI) could influence the virological response to first-line NNRTI therapy. OBJECTIVES: This study was designed to describe the proportions and quantities of NRTI and NNRTI-resistant variants in patients with successful first-line NNRTI therapy. STUDY DESIGN: We evaluated the presence of drug-resistance mutations (DRMs) prior to treatment initiation in 131 naive chronically HIV-1-infected patients initiating NNRTI-based first-line therapy. DRMs were detected by ultradeep pyrosequencing (UDPS) on a GS Junior instrument (Roche). RESULTS: The mean HIV RNA concentration was 4.78 ± 0.74 log copies/mL and the mean CD4 cell count was 368 ± 184 CD4 cells/mm(3). Patients were mainly infected with subtype B (68%) and 96% were treated with efavirenz. The sensitivity threshold for each mutation was 0.13-1.05% for 2000 reads. Major NRTI-resistant or NNRTI-resistant mutations were detected in 40 patients (33.6%). The median frequency of major NRTI-resistant mutations was 1.37% [IQR: 0.39-84.1], i.e.: a median of 556 copies/mL [IQR: 123-37,553]. The median frequency of major NNRTI-resistant DRMs was 0.78% [IQR: 0.67-7.06], i.e.: a median of 715 copies/mL [IQR: 391-3452]. The genotypic susceptibility score (GSS) of 9 (7.3%) patients with mutations to given treatment detected by UDPS was 1.5 or 2. CONCLUSIONS: First-line NNRTI-based treatment can produce virological success in naïve HIV-1-infected patients harboring low-frequency DRMs representing <1% of the viral quasispecies. Further studies are needed to determine the clinical cut-off of low-frequency resistant variants associated to virological failure.

Analytical sensitivity of three real-time PCR assays for measuring subtype B HIV-1 RNA.

BACKGROUND: Lack of HIV RNA during antiretroviral therapy (ART) is regarded as a desirable outcome. Commercial assays of HIV virus load now need to detect virus RNA concentrations below 50 c/ml and several of them have claimed a limit of detection (LOD) of 20-45 c/ml. OBJECTIVES AND STUDY DESIGN: We have compared the performances of three commercial assays of HIV RNA, the Abbott RealTime HIV-1, the Qiagen Artus RG HIV-1 and the Roche Cobas Ampliprep Cobas TaqMan (CAPCTM) HIV-1 vs 2.0 using replicate of specimens with HIV-1 subtype B RNA concentrations of 20-200 c/ml. RESULTS: Despite fair-to-moderate agreement between the three assays, probit analysis showed that their LODs differed; they were 81, 65 and 18c/ml respectively. The CAPCTM HIV-1 vs 2.0 values were higher than those of the other two; the maximum difference was 0.26 log c/ml. By testing 20 replicate of each concentration, coefficients of variation were between 0.6% and 9.2% (Abbott RealTime HIV-1), 10.3% and 38% (Qiagen Artus RG HIV-1) and 5.2% and 13.1% (Roche CAPCTM HIV-1 vs 2.0). The three assays also differed in their reproducibility and linearity for virus loads of 50-200 c/ml. CONCLUSION: The analytical performances of commercial virus load assays differ. Direct comparisons of widely used commercial assays in clinical studies could help to identify the residual viremia that is clinically relevant for effective long term therapy.

Minority variants associated with resistance to HIV-1 nonnucleoside reverse transcriptase inhibitors during primary infection.

BACKGROUND: Recent data suggest that subjects harbouring low-frequency variants of HIV that are resistant to non-nucleoside reverse-transcriptase inhibitors (NNRTI) could suffer virological failure when treated with NNRTI-based therapy. Rilpivirine, a second-generation NNRTI, will be used in first-line regimen therapy, but the prevalence of minority variants that are resistant to rilpivirine is unknown. OBJECTIVES: We evaluated the presence of low-frequency NNRTI resistance associated mutations (RAMs) in 27 patients with a primary HIV-1 infection. STUDY DESIGN: We performed genotypic resistance test at baseline and used ultradeep pyrosequencing (UDPS) to detect minority RAMs. RESULTS: Bulk genotyping identified NNRTI-resistant RAMs in 3/27 (11%) patients while UDPS identified NNRTI-resistant RAMs in 10/27 (37%) patients. The 11 RAMs not detected by bulk sequencing were A98G (n=2), L100I (n=3), K101E (n=2), V106I (n=3) and E138G (n=1). The prevalence of these minority variants was 0.34-18.26%. The absolute copy numbers of minority resistant variants were 3.21-5.53 log copies/mL. CRF02 harboured more minority resistant variants than subtypes B (P<0.05). Four samples (15%) had a major rilpivirine resistant mutation (E138G, K101E and E138A), 3 of which were detected by UDPS. CONCLUSION: In these primary HIV infected patients, as regards to the detection of RAMs at the cut-off level>15-25% of the virus population, the concordance between bulk genotypic and UDPS was perfect. UDPS detected additional major NNRTI-resistant mutations, including rilpivirine resistant variants. Further studies are needed to assess the impact of these minority variants on treatment efficacy.

Evaluation of the QIAsymphony™ SP and Artus™ RealTime extraction-quantification systems for measuring HIV-1 virus load.

BACKGROUND: Automated HIV-1 RNA extraction and its quantification by real-time PCR assays provide improved sample processing and better analytical performances. The new Artus HIV-1 RealTime assay can be performed after automated extraction with the Qiagen Qiasymphony robot. OBJECTIVES AND STUDY DESIGN: To evaluate the sensitivity, reproducibility, linearity, ability to detect HIV-1 subtypes of the Qiagen Qiasymphony and RealTime Artus HIV-1 assay system and to compare with the Roche Cobas Ampliprep Cobas TaqMan assay, vs 2.0 (CAP/CTM; Roche Molecular Systems). RESULTS: The detection limit calculated by probit analysis was 65 c/ml using dilutions of a NIBSC. Assays of serially diluted clinical samples gave very good inter-assay and intra-assay reproducibilities (<10% CV) and linearity (2.2-6.5 log copies/ml). The results Artus™ HIV-1 and CAP/CTM assays provided very similar results: average difference=0.11 log copies/ml. The Artus™ titers for 18 (22%) of the 114 HIV-1 group M samples tested differed by over 0.5 log copies/ml from the CAP/CTM titers. The Artus™ values were lower than the CAP/CTM values in 83% of cases. Discrepant results were not associated with particular subtypes. CONCLUSION: The Artus™ HIV-1 assay reliably quantified the HIV RNA in clinical specimens. Analytical performances were good and it integrated well with the Qiasymphony automated RNA extraction procedure. It appears to be appropriate for monitoring therapy and the routine management of HIV-1 infections.

Influence of HCV genotype 1 subtypes on the virus response to PEG interferon alpha-2a plus ribavirin therapy.

New factors that influence the viral response in HCV non-genotype 2/3 patients must be identified in order to optimize anti-HCV treatment. This multicenter prospective study evaluates the influence of HCV variability and pharmacological parameters on the virological response of these patients to pegylated interferon α2a (peg-IFN-α2a: 180 µg/week) and ribavirin (RBV; 800-1,200 mg/day) for 48 weeks. HCV subtypes were identified by sequencing the NS5B region. Serum RBV and peg-IFN-α2a concentrations were measured at weeks 4 and 12. The 115 patients (67 men; median age = 49, range 31-76) included 64 who had never been treated and 27 co-infected with HIV. The mean baseline HCV RNA was 6.30 ± 0.06 log IU/ml and the HCV genotypes were: G1 (n = 93) with 1a (n = 37) and 1b (n = 50), G4 (n = 20) and G5 (n = 2). Most patients (79/108; 73%) had an early virological response. Independent predictors of an early virological response were interferon naive patients (OR= 2.98, 95% CI: 1.15-7.72) and RBV of >2,200 ng/ml at week 12 (OR = 3.41, 95% CI: 1.31-8.90). Forty of 104 patients (38%) had a sustained virological response. The only independent predictors of a sustained virological response were subtype 1b (OR = 6.82, 95% CI: 1.7-26.8), and HCV RNA <15 IU/ml at week 12 (OR = 25, 95% CI: 6.4-97.6). Thus a serum RBV concentration of >2,200 ng/ml was associated with an early virological response and patients infected with HCV subtype 1b had a better chance of a sustained virological response than did those infected with subtype 1a.

Detection and quantitation of HCV RNA using real-time PCR after automated sample processing.

There is considerable evidence that the loss of hepatitis C virus (HCV) RNA during the first 3 months of treatment with pegylated interferon plus ribavirin is a prognostic marker of response to therapy. Real-time polymerase chain reaction (PCR) assays for quantifying HCV RNA in plasma or serum are now commercially available. The extraction of HCV RNA can also be automated. This report analyses the performance of the COBAS Ampliprep-COBAS Taqman 48 (CAP/CTM) real-time PCR assay and compares this new test with the COBAS Amplicor HCV Monitor v 2.0 assay (CAM). CAP/CTM was 100% specific. The assay was linear across a wide range of HCV RNA concentrations without sample dilution. The intra-assay variation was 0.3-3.3% and the interassay variation was 1.5-6.7%. A total of 118 clinical samples with different HCV genotypes were assayed using both methods. The results obtained using the two methods were well correlated (r = 0.89, P < 0.001). The mean difference [CAP/CTM-CAM] was 0.17 log IU/ml and it was not influenced by the HCV genotype or by the subtype. It is concluded that the new CAP/CTM system is adequate for quantifying HCV RNA in clinical practice.

Respiratory-related evoked potentials in children with asthma.

AIMS OF THE STUDY: Respiratory-related evoked potentials (RREPs) are a method of recording brain activities in response to respiratory stimuli. Although data in childhood are scarce, the absence of the early P1 component of RREPs has been reported in children with a history of life-threatening asthma. This study was focused on the presence, latencies, and amplitudes of the P1, N1, P2, and N2 components of the RREPs in a paediatric series of asthmatic patients. PATIENTS AND METHODS: RREPs were recorded in 21 patients with stable asthma, age range 8-17 years, 11 healthy children, age range 6-16 years, and 24 healthy adults, age range 20-28 years. The signals from left (C3-Cz) and right (C4-Cz) central (rolandic) location were recorded separately, using surface electrodes. Evoked responses to two series of 80 consecutive mid-inspiratory occlusions were averaged. Recordings were analysed manually. RESULTS: All 4 RREPs components were significantly more often absent in asthmatic children than in healthy children and adults (P1, p=0.01; N1, p=0.008; P2, p=0.008, N2, p=0.01). The latencies and amplitudes of the four components were similar in patients and healthy subjects. CONCLUSION: RREPs components were less frequently present in children with asthma than in healthy subjects. This finding should promote the recording of RREPs in other acute and chronic respiratory diseases in children in order to search for possible electroclinical correlations.

Sniff nasal inspiratory pressure: what is the optimal number of sniffs?

Sniff nasal inspiratory pressure (SNIP) measurement is a volitional noninvasive assessment of inspiratory muscle strength. A maximum of 10 sniffs is generally used. The purpose of the present study was to investigate whether the maximum SNIP improved after the tenth sniff. In total, 20 healthy volunteers and 305 patients with various neuromuscular and lung diseases were encouraged to perform 40 and 20 sniffs, respectively. The best SNIP among the first 10 sniffs was lower than the best SNIP among the next 10 sniffs in the healthy volunteers and patients. The SNIP improvement after the twentieth sniff was marginal. In conclusion, a learning effect persists after the tenth sniff. The current authors suggest using 10 additional sniffs when the best result of the first 10 sniffs is slightly below normal, or when sniff nasal inspiratory pressure is used to monitor a progressive decline in inspiratory muscle strength.

Pegylated interferon and ribavirin therapy for chronic hepatitis C virus genotype 4 infection.

Hepatitis C Virus (HCV) is classified into six genotypes. Genotype 4 is now spreading in Europe, especially among drug users, who are often infected with both HCV and the human immunodeficiency virus (HIV). Previous studies have shown that HCV-4 responds poorly to interferon. Pegylated interferon (peg-IFN) associated with ribavirin is now the most effective treatment for eradicating the virus. We have now studied the response of HCV-4 to peg-IFN and ribavirin and investigated the influence of HIV infection on anti-HCV therapy. Twenty-eight patients infected with HCV-4 were given peg-IFN plus ribavirin for 48 weeks. Patients infected with HCV alone tended to have a better initial response (66%) than patients infected with both HCV and HIV (30%, P = 0.06) and eradication was better (50%) than in doubly infected patients (15%, P = 0.06). After controlling for major factors influencing virus response, the virus response 12 weeks after the beginning of treatment in patients infected with HCV-4 (50%) was similar to that of patients infected with genotype 1 (53%) and lower than that of patients infected with genotypes 2 or 3 (82%, P < 0.05). The response 24 weeks after the end of therapy in patients infected with HCV-4 (32%) was similar to that of patients infected with HCV-1 (28%) and lower than that of patients with HCV-2 or HCV-3 (62% P < 0.05). These results indicate that HCV-4 patients should be considered to be difficult-to-treat.

Setting of noninvasive pressure support in young patients with cystic fibrosis.

The aim of the current study was to compare a clinical noninvasive method of setting up noninvasive pressure support ventilation (PS-NI) in young patients with cystic fibrosis (CF), based on parameters such as breathing frequency, arterial oxygen saturation and comfort rating, with a more invasive method (PS-I) targeted at optimising unloading of the inspiratory muscles and enhancing patient-ventilator synchronisation. PS-NI and PS-I were compared in random order in 10 children with CF. PS-NI differed from PS-I with regard to the level of inspiratory pressure (n=5), rate of inspiratory pressurisation (n=1), inspiratory trigger sensitivity (n=2) and expiratory trigger sensitivity (n=5). Although both methods modified breathing pattern, improved oxygen saturation and reduced diaphragmatic pressure time product (450+/-91 cmH2O.s(-1).min(-1) during spontaneous breathing, and 129+/-125 and 104+/-75 cmH2O.s(-1).min(-1) during PS-NI and PS-I, respectively), patient-ventilator synchrony and patient comfort were enhanced more during PS-I. In young patients with cystic fibrosis, setting up pressure support using a clinical noninvasive approach based on easily measurable parameters, such as respiratory rate and comfort rating, is as effective as a more invasive technique based on unloading of the inspiratory muscles and optimising patient-ventilator synchronisation. However, whilst the standard clinical method is satisfactory in the majority of patients, more invasive measurements should be considered in patients who have difficulty synchronising with the ventilator to enhance patient tolerance and compliance.

[C2 deficiency discovered in pneumococcal meningitis]. / Déficit en C2 découvert au décours d'une méningite à pneumocoque.

BACKGROUND: Congenital deficiencies of complement system proteins are rare. Patients with C2 deficiency have a high incidence of vascularitis syndromes. Most patients with this deficiency have no problems with increased susceptibility to infection, most commonly due to pneumococci, presumably because of the protective function of the alternative pathway. CASE REPORT: A 22 month-old girl was admitted because of acute meningitis and otitis. She had had 2 episodes of otitis media at the age of 1 year. Analysis of the CSF showed that this meningitis was due to pneumococcal infection. Recovery was complete after 15 days of antibiotic therapy. Total hemolytic complement activity (CH50) was low during the infection; one month later, the CH50 value was about zero as was C2, while C3 and C4 were normal. The patient was given polyvalent pneumococcal and anti-Haemophilus vaccines plus prophylactic penicillin G. Laboratory tests for systemic lupus erythematosus were negative. CONCLUSION: A defect of complement function should be suspected in any patient with severe of recurring pyogenic infections. Complement disorders can be detected one month later by means of the relatively simple hemolytic complement assay.