Recurrence of hepatitis C virus after treatment with pegylated interferon and direct acting antivirals in Punjab Pakistan

Although increased response rates concomitant in hepatitis C virus but relapse after treatment is threatened. Therefore, it is terrible requirement to evaluate the response of Pegylated interferon and direct acting antivirals in Punjab Pakistan. The study was conducted to find the rate of recurrence of HCV infection after treatment with Pegylated Interferon and Direct Acting Antivirals in Punjab Pakistan. This study was conducted at Department of Pathology, Nawaz Sharif Medical College Gujrat, while treatment effects monitored in different Government and Private Hospitals of Punjab, Pakistan. Total 973 patients who administered the recommended dose and divided in two groups (i) Interferon based therapy (ii) direct acting antivirals (DAAs).Other parameters like ALT and viral load studied. The rate of recurrence was higher in female infected with genotype 2b and in male with mixed genotype 3a/2b after six month of antiviral therapy. Genotype 3a showed significant response to therapy after three month. 32 among 374 (8.5%) were positive after 24 weeks of treatment with interferon, 29 (7.7%) patients have same genotype while 3 patients were re-infected with different HCV strains. With DAAs, only 27 (4.8%) patients were positive among 558 after 2 weeks and one patient re-infected with different genotype. Early and sustained virological response noted in DAAs. ALT and viral load decreased faster with DAAs that not achieved after 4 weeks with pegylated interferon. Sustained virological response appears in DAAs and recurrence rate is high in interferon therapy compared to DAAs. Therefore, reinfection has implications for correct treatment efficiency and to select strategies for retreatment cases.

Viral Infection Spreading and Mutation in Cell Culture

A new model of viral infection spreading in cell cultures is proposed taking into account virus mutation. This model represents a reaction-diffusion system of equations with time delay for the concentrations of uninfected cells, infected cells and viral load. Infection progression is characterized by the virus replication number Rv, which determines the total viral load. Analytical formulas for the speed of propagation and for the viral load are obtained and confirmed by numerical simulations. It is shown that virus mutation leads to the emergence of a new virus variant. Conditions of the coexistence of the two variants or competitive exclusion of one of them are found, and different stages of infection progression are identified.

Evaluation and modelling of the performance of an automated SARS-CoV-2 antigen assay according to sample type, target population and epidemic trends

The Lumipulse® G SARS-CoV-2 Ag assay performance was evaluated on prospectively collected saliva and nasopharyngeal swabs (NPS) of recently ill in- and outpatients and according to the estimated viral load. Performances were calculated using RT-PCR positive NPS from patients with symptoms ≤ 7 days and RT-PCR negative NPS as gold standard. In addition, non-selected positive NPS were analyzed to assess the performances on various viral loads. This assay yielded a sensitivity of 93.1% on NPS and 71.4% on saliva for recently ill patients. For NPS with a viral load > 103 RNA copies/mL, sensitivity was 96.4%. A model established on our daily routine showed fluctuations of the performances depending on the epidemic trends but an overall good negative predictive value. Lumipulse® G SARS-CoV-2 assay yielded good performance for an automated antigen detection assay on NPS. Using it for the detection of recently ill patient or to screen high-risk patients could be an interesting alternative to the more expensive RT-PCR.

Modelling upper respiratory viral load dynamics of SARS-CoV-2

Relationships between viral load, severity of illness, and transmissibility of virus are fundamental to understanding pathogenesis and devising better therapeutic and prevention strategies for COVID-19. Here we present within-host modelling of viral load dynamics observed in the upper respiratory tract (URT), drawing upon 2172 serial measurements from 605 subjects, collected from 17 different studies. We developed a mechanistic model to describe viral load dynamics and host response and contrast this with simpler mixed-effects regression analysis of peak viral load and its subsequent decline. We observed wide variation in URT viral load between individuals, over 5 orders of magnitude, at any given point in time since symptom onset. This variation was not explained by age, sex, or severity of illness, and these variables were not associated with the modelled early or late phases of immune-mediated control of viral load. We explored the application of the mechanistic model to identify measured immune responses associated with the control of the viral load. Neutralising antibodies correlated strongly with modelled immune-mediated control of viral load amongst subjects who produced neutralising antibodies. Our models can be used to identify host and viral factors which control URT viral load dynamics, informing future treatment and transmission blocking interventions.

“Facilitating HIV status adjustment: Qualitative insights from the Tambua Mapema proof-of-concept study in Kenya”

Systematic efforts are needed to prepare persons newly diagnosed with acute or chronic HIV infection to cope. We examined how patients dealt with this news, looking at how readiness to accept an HIV diagnosis impacted treatment outcomes, prevention of transmission, and HIV status disclosure. We examined vulnerability and agency over time and considered implications for policy and practice. A qualitative sub-study was embedded in the Tambua Mapema (“Discover Early”) Plus (TMP) study (NCT03508908), conducted in coastal Kenya between 2017 and 2020, which was a stepped wedge trial to evaluate an opt-out HIV-1 nucleic acid testing intervention diagnosing acute and chronic HIV infections. Diagnosed participants were offered antiretroviral therapy (ART), viral load monitoring, HIV partner notification services, and provision of pre-exposure prophylaxis (PrEP) to their uninfected partners. Data were analyzed using thematic approaches. Participants included 24 individuals who completed interviews at four time points (2 weeks and 3, 6, and 9 months after diagnosis), including 18 patients (11 women and 7 men) and 6 partners (1 woman, 5 men, of whom 4 men started PrEP). Acceptance of HIV status was often a long, individualized, and complex process, whereby participants’ coping strategies affected day-to-day issues and health over time. Relationship status strongly impacted coping. In some instances, couples supported each other, but in others, couples separated. Four main themes impacted participants’ sense of agency: acceptance of diagnosis and commitment to ART; positive feedback after attaining viral load suppression; recognition of partner supportive role and focus on sustained healthcare support whereby religious meaning was often key to successful transition. To support patients with acute or newly diagnosed chronic HIV, healthcare and social systems must be more responsive to the needs of the individual, while also improving quality of care, strengthening continuity of care across facilities, and promoting community support.

Maternal HIV Infection as a Risk Factor for Primary Epstein-Barr Virus Infection in Kenyan Infants

Human immunodeficiency virus (HIV) infection is known to be associated with EBV shedding in saliva suggesting an increased risk of EBV transmission to infants born to mothers with HIV at an earlier age. In this study we investigated (i) whether maternal HIV status was a risk factor for EBV in blood at delivery or for shedding in saliva and breast milk of 6- and 10-weeks post-partum mothers, (ii) if there was a difference in EBV strains shed between HIV+ and HIV- mothers, and (iii) if maternal HIV status was a determinant of EBV viral load in their infants. Samples were collected as part of a prospective cohort study that followed HIV-positive (HIV+) and HIV-negative (HIV-) pregnant women in Western Kenya through delivery and post-partum period. EBV viral load in blood was found to be significantly higher in mothers with HIV (p-value = 0.04). Additionally, a statistically significant difference was observed between EBV viral load in saliva samples and HIV status where HIV+ mothers had a higher EBV viral load in saliva at 6-weeks post-partum compared to HIV- mothers (p-value < 0.01). The difference in EBV shedding in breast milk was not found to be statistically significant. Furthermore, no difference in frequency of EBV strain was attributable to HIV- or HIV+ mothers. Interestingly, we found that infants born to HIV+ mothers had a higher EBV viral load at the time of their first EBV detection in blood than infants born to HIV- mothers and this was independent of age at detection. Overall, our study suggests that HIV infected mothers shed more virus in saliva than HIV-negative mothers and infants born to HIV+ mothers were at risk for loss of control of primary EBV infection as evidenced by higher EBV viral load following primary infection.

Comparison of Anterior Nares Viral Loads in Asymptomatic and Symptomatic Individuals Diagnosed with SARS-CoV-2 in a University Screening Program

RT-qPCR has been used as the gold standard method for detecting SARS-CoV-2 since early in the pandemic. At our university based high throughput screening program, we test all members of our community weekly. RT-qPCR cycle threshold (CT) values are inversely proportional to the amount of viral RNA in a sample, and thus are a proxy for viral load. We hypothesized that CT values would be higher, and thus the viral loads at the time of diagnosis would be lower in individuals who were infected with the virus but remained asymptomatic throughout the course of the infection. We collected the N1 and N2 CT values from 1633 SARS-CoV-2 positive RT-qPCR tests of individuals sampled between August 7, 2020, and March 18, 2021, at the BU Clinical Testing Laboratory. We matched this data with symptom reporting data from our clinical team. We found that asymptomatic patients had CT values significantly higher than symptomatic individuals on the day of diagnosis. Symptoms were followed by the clinical team for 10 days post the first positive test. Within the entire population, 78.1% experienced at least one symptom during surveillance by the clinical team (n=1276/1633). Of those experiencing symptoms, the most common symptoms were nasal congestion (73%, n=932, 1276), cough (60.0%, n=761/1276), fatigue (59.0%, n=753/1276), and sore throat (53.1%, n=678/1276). The least common symptoms were diarrhea (12.5%, n=160/1276), dyspnea on exertion (DOE) (6.9%, n=88/1276), foot or skin changes (including rash) (4.2%, n=53/1276), and vomiting (2.1%, n= 27/1276). Presymptomatic individuals, those who were not symptomatic on the day of diagnosis but became symptomatic over the following 10 days, had CT values higher for both N1 (median= 27.1, IQR 20.2- 32.9) and N2 (median=26.6, IQR 20.1-32.8) than the symptomatic group N1 (median= 21.8, IQR 17.2- 29.4) and N2 (median= 21.4, IQR 17.3- 28.9) but lower than the asymptomatic group N1 (median=29.9, IQR 23.6-35.5) and N2 (median= 30.0, IQR 23.1- 35.7). This study supports the hypothesis that viral load in the anterior nares on the day of diagnosis is a measure of disease intensity at that time.

Determinants of Therapy Failure Among Adults on First-line Antiretroviral Therapy in Asmara, Eritrea: A Multicenter Retrospective Matched Case-control Study

Background Information on treatment failure (TF) in People living with HIV in data-poor jurisdictions is necessary to counter the rapidly escalating epidemic of TF to first-line combined anti-retroviral therapies (cART) in sub-Saharan Africa (SSA). In this study, we examined the risk factors associated with TF in Asmara, Eritrea.Methods: A multicenter, retrospective 1:2 matched (by age and gender) case-control study was conducted in four major hospitals in Asmara, Eritrea on adults aged >15 years who were on treatment for at least 6 months. Cases were patients with viral load ≥1000 copies/mL anytime between 2019-2021 and/or patients switched to second line cART. Controls were randomly selected from patients on first-line ART with viral load < 1000 copies/mL. Data was extracted using a checklist from the master data set and analyzed using SPSS version 26. Multivariable logistic regression analysis was conducted to identify risk factors for TF. All p-values were 2-sided and the level of significance was set at p < 0.05 for all analyses.Results: Of the 1068 participants, 585 (54.7%) were females. The median age at treatment initiation was 46 years (interquartile range (IQR): 39–51). Median time to combined antiretroviral therapy (cART) failure was 37 months (IQR =24–47). In multivariate analysis factors associated with increased likelihood of virologic failure (VF) were the type of initially used nucleoside reverse transcriptase inhibitors (NRTI) backbone ( (Zidovudine+Lamivudine (AZT+3TC): adjusted odds ratio (aOR): 2.70; 95% Confidence interval (CI): 1.65-4.41, p-value<0.001), (Abacavir+lamivudine (ABC+3TC): aOR: 4.73; 95%CI: 1.18-18.92, p-value=0.028), and (Stavudine+Lamivudine (D4T+3TC): aOR: 5.00; 95% CI: 3.03-8.20, p-value<0.001), prior exposure to ART (aOR: 2.28; 95%CI:1.35–3.86; p=0.002), record of sub-optimal drug adherence (aOR: 3.08; 95%CI: 2.22–4.28; p<0.001), ambulatory/bedridden at presentation (aOR:1.61; 95%CI: 1.12-4.28; p-value=0.010), presence of comorbidities (aOR: 2.37; 95%CI: 1.36-4.10, p-value=0.002), duration of cART (<5 years: aOR: 5.90; 95% CI: 3.95-8.73, p-value<0.001), and use of SMX-TMP prophylaxis ( aOR : 2.00, 95%CI, 1.44-2.78, p-value<0.001). Conclusion: Our findings underscore the importance of optimizing cART adherence, diversification of cART regimens, and interventions directed at enhancing early HIV diagnosis, prompt initiations of treatment and improved patient focused monitoring of treatment response.

Infectious viral load in unvaccinated and vaccinated patients infected with SARS-CoV-2 WT, Delta and Omicron

Background Viral load (VL) is one determinant of secondary transmission of SARS-CoV-2. Emergence of variants of concerns (VOC) Alpha and Delta was ascribed, at least partly, to higher VL. Furthermore, with parts of the population vaccinated, knowledge on VL in vaccine breakthrough infections is crucial. As RNA VL is only a weak proxy for infectiousness, studies on infectious virus presence by cell culture isolation are of importance. Methods We assessed nasopharyngeal swabs of COVID-19 patients for quantitative infectious viral titres (IVT) by focus-forming assay and compared to overall virus isolation success and RNA genome copies. We assessed infectious viral titres during the first 5 symptomatic days in a total of 384 patients: unvaccinated individuals infected with pre-VOC SARS-CoV-2 (n= 118) or Delta (n= 127) and vaccine breakthrough infections with Delta (n= 121) or Omicron (n=18). Findings Correlation between RNA copy number and IVT was low for all groups. No correlation between IVTs and age or sex was seen. We observed higher RNA genome copies in pre-VOC SARS-CoV-2 compared to Delta, but significantly higher IVTs in Delta infected individuals. In vaccinated vs. unvaccinated Delta infected individuals, RNA genome copies were comparable but vaccinated individuals have significantly lower IVTs, and cleared virus faster. Vaccinated individuals with Omicron infection had comparable IVTs to Delta breakthrough infections. Interpretation Quantitative IVTs can give detailed insights into virus shedding kinetics. Vaccination was associated with lower infectious titres and faster clearance for Delta, showing that vaccination would also lower transmission risk. Omicron vaccine breakthrough infections did not show elevated IVTs compared to Delta, suggesting that other mechanisms than increase VL contribute to the high infectiousness of Omicron. Funding This work was supported by the Swiss National Science Foundation 196644, 196383, NRP (National Research Program) 78 Covid-19 Grant 198412, the Fondation Ancrage Bienfaisance du Groupe Pictet and the Fondation Privée des Hôpitaux Universitaires de Genève.

Quantitative Isothermal Amplification on Paper Membranes using Amplification Nucleation Site Analysis

Quantitative nucleic acid amplification tests (qNAATs) are critical in treating infectious diseases, such as in HIV viral load monitoring or SARS-CoV-2 testing, in which viral load indicates viral suppression or infectivity. Quantitative PCR is the gold standard tool for qNAATs; however, there is a need to develop point-of-care (POC) qNAATs to manage infectious diseases in outpatient clinics, low- and middle-income countries, and the home. Isothermal amplification methods are an emerging tool for POC NAATs as an alternative to traditional PCR-based workflows. Previous works have focused on relating isothermal amplification bulk fluorescence signals to input copies of target nucleic acids for sample quantification with limited success. In this work, we show that recombinase polymerase amplification (RPA) reactions on paper membranes exhibit discrete fluorescent amplification nucleation sites. We demonstrate that the number of nucleation sites can be used to quantify HIV-1 DNA and RNA in less than 20 minutes. An image-analysis algorithm quantifies nucleation sites and determines the input nucleic acid copies in the range of 67-3,000 copies per reaction. We demonstrate a mobile phone-based system for image capture and onboard processing, illustrating that this method may be used at the point-of-care for qNAATs with minimal instrumentation.