Heterogeneity and Hierarchy of Immune Response to Primary Immunization in HIV-Infected Children on HAART and the Impact of an Additional Dose of Vaccine.

The nature of vaccine response inferiority is not well studied in children living with HIV (CLHIV). The authors investigated Hepatitis B Virus (HBV) and Diphtheria/Pertussis/Tetanus toxoid (DPT) vaccination responses following primary immunization in CLHIV (n = 42) and healthy controls (HC) (n = 38) and the effect of an additional vaccine dose. Antibody responses, CD4 and HBV-specific T/B cells were analysed using CMIA/ELISA and flow-cytometry. CLHIV had significantly lower baseline median antibody titres for all vaccines than HC (p <0.02). Differential seroprotection rates observed in CLHIV were, 4.8% for pertussis; 9.5% for HBV; 26.2% for diphtheria and 66.7% for tetanus. WHO staging significantly influenced anti-HBs levels (p = 0.0095). HBsAg-specific CD4+T-cells were significantly higher in CLHIV than HC (p = 0.042). An additional vaccine dose (HBV and Tdap) conferred a higher protection rate for tetanus and diphtheria (p <0.040) in CLHIV. These findings suggest that CLHIV exhibit a hierarchy of vaccine responses affecting antibody levels and protection rate, which was rescued by administering additional vaccine dose.

Challenges and Pragmatic Solutions for Assessing the Reliability of HIV-1 Viral Load Monitoring in Resource-Constrained Settings.

INTRODUCTION: HIV-1 RNA detection is the most reliable method for monitoring treatment response among people living with HIV. Effective quality control measures that include internal quality control (IQC) are challenging in resource-constrained settings. METHODS: We ascertained the utility of the kit low positive control (LPC) as an effective IQC to monitor the reliability of the HIV-1 viral load assay. Variations in LPC values were measured for 390 different runs over 10 years (2011-2021) and compared to in-house IQC data using Levey-Jennings control chart. RESULTS: Overall, the Levey-Jennings analysis showed minimal variation (±0.5 log) for both the LPC and IQC data. The mean LPC value for first 20 runs (20 days) was 2.91. The mean LPC value for the 390 runs comprising 35 different lots was 3.01 ± 0.1 log. CONCLUSION: Our decadal data reveal that Abbott RealTime HIV-1 assay (Abbott Molecular Inc., IL, USA) LPC exhibited no significant biological variation over 390 runs distributed over 10 years. Hence, assay LPC can supplant the IQC for monitoring assay trends as a stable and commutable material in resource-constrained settings.

ARCHITECT HBsAg Next assay is positioned better to resolve and refine challenging weak reactive clinical samples.

BACKGROUND: Ultrasensitive HBsAg assays are replacing the previous versions. Unlike the sensitivity, the specificity, and its positioning to resolve weak-reactives (WR) are not studied. We investigated the ability of ARCHITECT HBsAg-Next (HBsAg-Nx) assay to resolve WR and sought its clinical validation and correlation with confirmatory/reflex testing. METHODS: Among 99,761 samples between Jan 2022 - 2023, 248 reactive samples in HBsAg-Qual-II were compared with HBsAg-Nx assay. Sufficient samples were further subjected to neutralization (n = 108) and reflex (anti-HBc total/anti-HBs antibody) testing. RESULTS: Out of 248 initial reactive samples in HBsAg-Qual-II, 180 (72.58%) were repeat reactive, and 68 (27.42%) were negative, whereas in HBsAg-Nx, 89 (35.89%) were reactive and 159 (64.11%) were negative (p<0.0001). Comparing the results of two assays (Qual-II/Next), 57.67% (n = 143) were concordant (++/-) and 105 (42.33%) were discordant (p = 0.0025). Testing of HBsAg-Qual-II + & HBsAg-Nx - samples revealed that 85.71% (n = 90) were anti-HBc total negative and 98.08% (n = 51) were not neutralized as well as significant proportion (89%) had no clinical correlation. The proportion of samples neutralized was significantly different between ≤5 S/Co (26.59%) and >5 S/Co (71.42%) (p = 0.0002). All samples (n = 26) with enhanced reactivity in HBsAg-Nx were effectively neutralized, while samples with no increase in reactivity, 89% (n = 72) failed neutralization (p=<0.001). CONCLUSIONS: HBsAg-Nx assay is positioned better to resolve and refine challenging WR samples than Qual-II which correlated well with confirmatory/reflex tests and clinical disease. This superior internal benchmarking significantly reduced the cost and quantum of retesting, confirmatory/reflex testing in the diagnosis of HBV infection.

Longitudinal assessment of HCV core antigen kinetics to monitor therapeutic response in the age of DAAs.

BACKGROUND: In the economy of therapeutic monitoring, an affordable viral marker is essential in the era of direct-acting antivirals (DAAs). We elucidated the kinetics of HCVcAg to delineate its precise role in monitoring therapeutic response. METHODS: In this longitudinal study, 3208 patients were tested for HCV RNA. A total of 423 patients were started on DAAs. Treatment response and kinetics of HCVcAg/RNA were assessed in treatment-naïve (n = 383) and previously treated (n = 40) patients with follow-up for 2 years. RESULTS: After the initiation of DAAs, the rate of relapse was significantly higher in the previously treated group than naive group [12.5% (5/40) Vs 2% (7/383), p<0.0001]. The response rate at RVR was significantly higher with HCVcAg than RNA in both groups (p<0.02). The kinetics of HCVcAg and RNA were significantly different at ETR and SVR12 in the naïve (p<0.04), but similar at all therapeutic points in the previously treated group. The correlation between HCVcAg and RNA was good at baseline, ETR and SVR, except RVR in both groups (r>0.6; p<0.0001). Furthermore, HCV genotypes, treatment regimen, CTP (<7/≥7) and MELD (<15/≥15) did not influence the therapeutic response and the viral replication kinetics (p>0.05). CONCLUSIONS: It is the first longitudinal study from India shows that the response rate and kinetics of HCVcAg are comparable to HCV RNA for an extended duration, except at RVR, irrespective of the HCV genotypes, treatment regimen, and liver disease severity. Hence, HCVcAg can be considered as a pragmatic marker to monitor therapeutic response and predict relapse in the era of DAAs.

Versatile performance edges of HBsAg Next assay in diagnosis and therapeutic monitoring of HBV infection.

BACKGROUND: HBsAg Next assay (HBsAgNx) claims improved detection of HBsAg. The aim was to investigate its performance in ascertaining HBsAg loss, ability to detect HBsAg in various phases of HBV infection, specificity and its amenability to in-house neutralization. METHODS: Analytical sensitivity was investigated using NIBSC standard (3rd WHO-IS). For clinical performance, out of 91,962 samples tested for HBsAg (Qual-II), 512 samples consisting of 170 cases with evidence of HBsAg loss during treatment (n = 116) and without treatment (n = 54), acute-hepatitis B (n = 90) and acute exacerbation of chronic-hepatitis B (n = 41), acute-hepatitis A (n = 24) and acute-hepatitis E (n = 9) positive, HIV-1 positive (n = 20), non-HBV, HAV and HEV related acute-hepatitis (n = 81) and HBsAg prozone (n = 14) as well as in-house neutralization (n = 63) were included. RESULTS: The calculated limit of detection (LOD) was 0.004 IU/mL. Of the 170 patients with apparent HBsAg loss, 18/116 (15.5%) among treated and 15/54 (27.7%) with spontaneous clearance were positive in HBsAgNx (p < 0.0001). Additionally, it detected HBsAg in 12/95 (12.6%) and 6/34 (17.6%) patients who were HBV DNA negative in treatment experienced and spontaneous clearance groups respectively (p < 0.001). The specificity of HBsAgNx was comparable to HBsAg Qual-II. The signal-intensity of HBsAgNx was significantly higher than HBsAg Qual-II across various phases of HBV infection and prozone samples. CONCLUSION: HBsAgNx significantly enhanced the accuracy of HBsAg detection without compromising the specificity in ascertaining HBsAg loss. The performance was superior in various phases of HBV infection including samples that exhibited prozone effect. Furthermore, it is amenable to cost-effective in-house neutralization to confirm low HBsAg levels.

Can In-house HIV-2 Viral Load Assay be a Reliable Alternative to Commercial Assays for Clinical and Therapeutic Monitoring?

BACKGROUND: Currently, there is a global contemplation to end the AIDS epidemic by 2030. HIV-2 poses unique challenges to this end. The burden of HIV-2 is higher in resource-limited countries, and it is intrinsically resistant to NNRTI drugs. In addition, there is no FDA-approved plasma viral load assay to monitor disease progression and therapeutic efficacy. To overcome these challenges, we have developed and evaluated an in-house quantitative HIV-2 viral load assay. METHODS: Blood samples were collected from 28 HIV-2 treatment-naïve monoinfected individuals and tested using an in-house qPCR HIV-2 viral load assay. The extracted RNA was amplified using Quantifast pathogen + IC kit. RESULTS: The in-house qPCR has a limit of detection of 695 copies/ml. The intra- and inter-assay variation (% CV) of the assay was 0.61 and 0.95, respectively. The in-house assay quantified HIV-2 NIBSC accurately (1000 IU) with a mean of 1952 copies/mL. Among the 28 samples tested by in-house qPCR assay, 11 (39.2%) samples were quantified, whereas 17 (60.7%) samples were not detected. In comparison with Altona RealStar HIV-2 RT PCR and Exavir Load RT assay, the results were 96.4% and 69.6% concordant, respectively. No significant (p = 0.99 and p = 0.13) difference in quantifying viral load between the three assays. Based on clinical and immunological (CD4) staging, the performance characteristics were comparable. CONCLUSION: To the best of our knowledge, this is the first in-house qPCR developed in India. The performance characteristics of the in-house assay are comparable to the commercial assays, and they can be used assertively to monitor HIV-2 patients.

Chronic Immune Activation Among Treatment Naïve HIV/ HBV Coinfected Individuals From Southern India.

BACKGROUND: Chronic immune activation is one of the most widely recognized hallmarks of HIV infection. T-cells that express CD38+ and HLA-DR+ show poor proliferative potential, signal transduction, and increased apoptotic potential. This affects HIV pathogenesis and its outcome and further complicates with a coinfection like HBV. METHODS: Study Design: cross-sectional. Blood samples were collected and analyzed for virological markers using ELISA for HBeAg and RT-PCR for HIV&HBV Viral load. Chronic immune activation markers of CD8+ and CD4+ T cells were measured by Flow cytometry for both HIV and HBV. RESULTS: There was a significant increase in HBV replication shown by higher HBV DNA (p=0.002), a higher proportion of HBeAg (p=0.0049), and lower CD4 counts (p=0.04) among HIV/HBV coinfected individuals, compared to the monoinfected groups. The frequencies of CD4+ CD38+ HLA-DR+ and CD8+ CD38+ HLA-DR+ in the HIV/HBV coinfection were significantly higher than HBV monoinfected group (P< 0.0001) and in the HIV monoinfected group (P < 0.0001). The Liver fibrosis score APRI and FIB-4, were higher in the coinfected group compared with HBV monoinfected group (0.67 vs. 0.25, p = 0.0085; 3.48 vs. 0.98, p = 0.0026) respectively. The cytokine levels of IL-17, Fas-L,TNF -α, IL-10, IL-2 and Granzyme B were also measured and compared among the study groups. CONCLUSION: Our data suggest that HIV probably influences immune activation of CD4+ and CD8+ T cells and this may play a significant role in accelerating the disease outcome among HIV/HBV coinfected individuals.

Clinical utility of hepatitis C virus core antigen (HCVcAg) assay to identify active HCV infection in hemodialysis and renal transplant patients.

BACKGROUND: The prevalence of HCV infection is high and it is a major cause of liver-related morbidity and mortality in hemodialysis and renal transplant patients. Diagnosis of hepatitis C virus (HCV) infection requires both HCV antibody screening and confirmatory nucleic acid testing (NAT). Hepatitis C virus core antigen (HCVcAg) is a reliable direct viral marker to identify active HCV infection. AIM: To assess the clinical utility of HCV core antigen to identify active HCV infection in hemodialysis and renal transplant patients. METHODS: A representative total of 231 plasma samples with a predominance of low viral load were included for HCVcAg testing and its performance characteristics were compared with the gold standard HCV RNA. RESULTS: Comparison of HCVcAg with HCV RNA showed an excellent specificity of 99% (95% CI: 94.7 to 100%) and sensitivity of 80.62% (95% CI: 73.59 to 87.7%). Likewise, the PPV and NPV of HCVcAg were 99.1% (95% CI: 93.7% to 99.9%) and 80.2% (95% CI: 74% to 85.2%) respectively. The correlation between HCVcAg and HCV RNA was found to be good (R2 = 0.86, p<0.0001). Among common Indian HCV genotypes (1, 3 & 4), good correlation was observed between HCV RNA and HCVcAg (R2 = 0.81, p <0.0001). CONCLUSIONS: It is the first Indian study to show that HCVcAg is a reliable, cost-effective direct marker to identify active HCV infection in hemodialysis and renal transplant patients. Implementation of HCVcAg testing could improve the accessibility to efficacious and affordable disease management in hemodialysis and renal transplant patients. In HCVcAg negative cases, sequential testing with anti-HCV antibody followed by HCV RNA could be a reliable and cost-effective approach.

Characteristics of treatment-naïve HBV-infected individuals with HIV-1 coinfection: A cross-sectional study from South India.

Purpose: Human immunodeficiency virus-1 (HIV-1) and hepatitis B virus (HBV) coinfection has become a major health problem across the globe. The increased life expectancy of HIV-1 patients due to antiretroviral therapy has led to the emergence of liver disease as a major mortality factor among them. The purpose of the study was to examine the baseline characteristics of HBV in treatment-naïve HBV/HIV coinfection from southern India compared to monoinfected individuals. Materials and Methods: The study was cross sectional in design, and samples were examined from 80 HIV-1, 70 HBV and 35 HBV/HIV-coinfected individuals using chemiluminescent microparticle immunoassay, real-time polymerase chain reaction and flow cytometry assays. Results: There was a significant increase in HBV DNA (P = 0.0001), higher hepatitis B e antigen percentage difference (P = 0.027) and lower CD4 counts (P = 0.01) among the HBV/HIV-coinfected individuals, but no difference in the HIV-1 viral load compared to HIV-1-monoinfected individuals. Also, the aspartate aminotransferase levels, prothrombin time and the international normalised ratio were significantly high among coinfected individuals. Conclusion: These findings conclude that HIV-1 coinfection can have serious implications on the outcome of HBV-related liver disease. To the contrary, HBV infection had no consequence on the progression of HIV-1 disease but distinctly lowered CD4+ T-cells.

Evaluation of dried blood spots as a feasible alternative to plasma for the detection and quantification of hepatitis c virus in a tropical setting: A pilot study.

Introduction: Confirmatory diagnosis of hepatitis C virus (HCV) infection (HCV RNA detection) is essential before start of the therapy. HCV RNA detection is not available in many parts of India. Shipment of plasma from distant places to referral laboratories may affect HCV RNA titres. Dried blood spots (DBS) provide an easy alternative for transporting samples to centres where HCV RNA testing is done. Aim: Evaluation of DBS as a feasible alternative to plasma for HCV diagnosis. Methods: In this cross-sectional study, 40 consecutive patients' blood samples were collected from patients referred from the Liver Clinic. Whole blood was spotted onto two Whatman 903TM cards. One card was incubated at ≥37°C and other at 4°C for 15 days, after drying. DBS was eluted and run in Abbott RealTime HCV assay. HCV was also quantified using the Abbott ARCHITECT HCV core antigen assay for 29 of the study patients. Results were compared with normal plasma values. Results: The median log HCV RNA value (in log10IU/mL) of plasma was 5.74, with normalised DBS it was 4.92 (≥37°C) and 4.66 (4°C); difference in plasma and DBS median log values was 0.82 (≥37°C) and 1.08 (4°C) logs, respectively. Interclass correlation values were 0.943, P < 0.0001 (≥37°C) and 0.950, P < 0.0001 (4°C), showing high agreement. The median HCV core antigen value (in fmol/L) for plasma was 325.35, whereas it was 4.77 (≥37°C) and 4.64 (4°C) for DBS samples. Conclusions: DBS can be used for sampling patients from distant resource-limited settings as an alternative to plasma for HCV RNA estimation. Larger studies are required to evaluate the feasibility of DBS in the Indian subcontinent, especially for HCV core antigen estimation.

Performance of a Modified In-House HIV-1 Avidity Assay among a Cohort of Newly Diagnosed HIV-1 Infected Individuals and the Effect of ART on the Maturation of HIV-1 Specific Antibodies.

BACKGROUND: Viral kinetics impact humoral immune response to HIV; antibody avidity testing helps distinguish recent (<6 months) and long-term HIV infection. This study aims to determine the frequency of recent HIV-1 infection among clients attending ICTC (Integrated Counselling and Testing Centre) using a commercial EIA, to correlate it with a modified in-house avidity assay and to study the impact of ART on anti-HIV-1 antibody maturation. METHODS: Commercial LAg Avidity EIA was used to detect antibody avidity among 117 treatment naïve HIV-1 infected individuals. A second-generation HIV ELISA was modified for in-house antibody avidity testing and cutoff was set based on Receiver Operating Characteristic (ROC) analysis. Archived paired samples from 25 HIV-1 infected individuals before ART and after successful ART; samples from 7 individuals responding to ART and during virological failure were also tested by LAg Avidity EIA. RESULTS: Six individuals (5.1%) were identified as recently infected by a combination of LAg avidity assay and HIV-1 viral load testing. The modified in-house avidity assay demonstrated sensitivity and specificity of 100% and 98.2%, respectively, at AI=0.69 by ROC analysis. Median ODn values of individuals when responding to ART were significantly lower than pre-ART [4.136 (IQR 3.437- 4.827) vs 4.455 (IQR 3.748-5.120), p=0.006] whereas ODn values were higher during virological failure [4.260 (IQR 3.665 - 4.515) vs 2.868 (IQR 2.247 - 3.921), p=0.16]. CONCLUSION: This modified in-house antibody avidity assay is an inexpensive method to detect recent HIV-1 infection. ART demonstrated significant effect on HIV-1 antibody avidity owing to changes in viral kinetics.

Evaluation of reliability and performance of hepatitis B virus-e-antigen assays in tertiary care setting.

Hepatitis B virus-e-antigen (HBeAg) is a viral marker to assess hepatitis B virus (HBV) replication. We have evaluated the reliability of three commonly available HBeAg immunoassays using World Health Organization-International Standard and clinical samples. In addition the performance of enzyme immunoassays (EIAs) was assessed by kinetic binding and reagent exchange experiments. Analytical and diagnostic sensitivity were significantly different among HBeAg assays (P < 0.01). The affinity of capture/detector antibodies varied significantly between EIAs (P < 0.01). Our findings suggest that significant difference in the affinity of capture/detector antibodies to HBeAg may impact the overall performance and the reliability of currently available HBeAg assays in HBV diagnosis and management.

Diagnostic reliability of Architect anti-HCV assay: Experience of a tertiary care hospital in India.

BACKGROUND & AIMS: Anti-HCV assays are prone to false positive results. Thus, accurate detection of HCV infection is critical for the timely therapeutic management. This study ascertained the reliability of Architect anti-HCV assay (Abbott) and to estimate the agreement of this assay with Ortho HCV 3.0 ELISA Test System with Enhanced SAVe (Ortho), HCV Tri-dot (Tri-dot) and HCV-PCR in a tertiary care setting. METHODS: A total of 78 788 consecutive sera were routinely screened for anti-HCV antibodies using Architect. All repeatedly reactive anti-HCV sera (n=1000) and anti-HCV negative sera (n=300) were tested in Ortho and in Tri-dot assays. Representative proportions of sera (n=500) with various signal-to-cut-off (S/Co) ratio were also compared with HCV-PCR. RESULTS: When Architect was compared with Ortho, Tri-dot, and HCV-PCR, the level of agreement as assessed by kappa were .26, .16, and .27 respectively. Using Latent class analysis (LCA), we found that sensitivity and specificity were 100% and 36.1% for Architect, 93.8% and 100% for Ortho and 63.8% and 100% for Tri-dot respectively. The median S/CO ratio of Architect and Ortho anti-HCV assays were significantly different between HCV-PCR positive and negative results (P<.0001). Furthermore, Architect S/CO ratio of >8 showed higher accuracy indices in both anti-HCV assays. CONCLUSIONS: Architect can be used as a screening assay because of its high sensitivity, high throughput, and short turnaround time. However, S/Co ratios of ≥1 to <8 in Architect necessitates HCV PCR to identify current infection and or EIA to distinguish true positivity from false biological positivity.

Cost-Effective In-House Neutralization Assay for the Confirmation of HBeAg.

BACKGROUND & AIM: Hepatitis B virus-e-antigen (HBeAg) is an affordable viral marker to assess viral replication kinetics and response to antiviral therapy. In the absence of confirmatory assays, discrepant or false-positive HBeAg results are resolved by screening for other HBV markers. We standardized an in-house HBeAg neutralization assay (HBeAg-NT) to confirm HBeAg in clinical samples. METHODS: The performance and reliability of this assay were evaluated by first WHO International Standard for HBeAg (first WHO-IS HBeAg) from Paul Ehrlich Institute and clinical samples (n = 150) from chronic HBV carriers. Of these, 71 HBeAg-positive sera were used for HBeAg-NT. RESULTS: Concentrations spanning 0.25-10 U of first WHO-IS HBeAg and clinical samples (S/Co ranges from 1.00 to 10.00) were neutralized completely in the HBeAg-NT. CONCLUSIONS: HBeAg-NT is a simple, cost-effective, and reliable direct approach to confirm HBeAg in clinical samples which precludes the need for screening additional HBV markers in low resource settings.

Serum HBsAg quantification in treatment-naïve Indian patients with chronic hepatitis B.

BACKGROUND AND AIMS: There is paucity of Indian data regarding serum HBsAg levels (qHBsAg) in treatment-naïve chronic hepatitis B (CHB). This study was done to determine correlation of qHBsAg with hepatitis B e antigen (HBeAg) and hepatitis B virus (HBV) DNA levels and its ability to independently categorize subgroups of CHB. METHODS: We studied 131 treatment-naive CHB patients and initially classified them based on HBeAg status. The HBeAg-positive group was further classified into immune tolerance (IT) and immune clearance (IC) phases based on serum alanine aminotransferase. HBeAg-negative patients were classified into low replicators (LR) and HBeAg-negative chronic hepatitis (ENH) based on DNA levels. HBsAg quantification was performed using the Architect chemiluminescence system. RESULTS: HBeAg-positive patients had higher DNA (7.89 vs. 2.69 log10 IU/mL) and higher qHBsAg (4.60 vs. 3.85 log10 IU/mL) compared to the HBeAg-negative group. Good correlation between qHBsAg and DNA was seen in HBeAg-positive (ρ = 0.6, p < 0.001) but not in HBeAg-negative CHB (ρ = 0.2). A qHBsAg level greater than 4.39 log10 IU/mL predicted HBeAg-positive state with 81 % sensitivity and 85 % specificity. However, among HBeAg-negative CHB, qHBsAg failed to discriminate between LR and ENH. CONCLUSIONS: A single point estimation of qHBsAg in treatment-naïve patients could predict replicative HBeAg-positive CHB, but was not helpful in defining replicative status in the HBeAg-negative CHB.

Why does size matter for viruses--a new paradigm on viral size.

Size and shape are the two immutable laws that govern all life forms including viruses. In this study we postulate and evaluate the hypothesis that there exists a strong association between viral geometry and features of viral disease outbreaks. Data on viral disease outbreaks were retrieved from WHO and CDC public domains for a period of twelve years to assess the relationship between viral size and epidemiological factors such as number of outbreaks, case fatality rate, proportion of emerging infectious diseases and transmission routes. We observed a significant correlation between viral size and frequency of disease outbreaks (rho=-0.82, p=0.004), case fatality rate (rho=0.48, p=0.03) and genome size (r=0.79, p<0.001). Viral sizes were significantly different among diverse transmission routes (p<0.001). The proportion of emerging infectious diseases were significantly different between viruses with size <10(5) and >or=10(5)nm(3) (21% vs 64%, p=0.046). In conclusion, this preliminary evidence shows that viral size plays a substantial role in the epidemiology of viral diseases. Our data suggests that small size viruses are associated with more number of outbreaks than large size viruses. Large size viruses are associated with high case fatality rate and can be potential emerging pathogens. Viral size may be crucial for niche selection and specified transmission routes in the susceptible host. Hence, viral geometry should not be neglected in epidemiology and modeling of viral diseases, and planning vaccine strategies.