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INTRODUCTION: Human pegivirus-1 (HPgV-1) influences the pathogenesis and outcome of viral infections. We investigated the prevalence and impact of HPgV-1 due to the paucity of studies on Indian people living with HIV (PLHIV). METHODOLOGY: Samples were collected from 347 treatment-naïve PLHIV; and 100 blood donors negative for HIV, HBV, and HCV. CD4+ T-cell and HIV-1 viral load were measured using flow-cytometry and quantitative polymerase chain reaction (qPCR), respectively. HPgV-1 was quantified and genotyped by qPCR and Sanger sequencing, respectively. RESULTS: HPgV-1 viremia in PLHIV and controls was 11% (38/347) and 1% (1/100), respectively. We found HPgV-1 genotype-2a in PLHIV and genotype-2b in controls. Male preponderance was seen in HIV-1 mono-infection and co-infection groups (166 vs. 143 and 33 vs. 5; p < 0.0001). The peak prevalence of HPgV-1 was at 31-50 years (p = 0.02). CD4+ T-cell count (245.5 vs. 240; p = 0.59) and HIV-1 log viral load (4.7 vs. 4.9; p = 0.50) were not significantly different between the HIV-1 mono-infected and coinfected individuals. However, a direct correlation existed between HpgV-1 viral load and CD4+ T-cell count (r = 0.27, p = 0.05) and an inverse correlation with HIV-1 viral load (r = -0.21, p = 0.10). CONCLUSIONS: This is the first study in India to estimate the HPgV-1 prevalence in PLHIV with the predominance of genotype-2a. HPgV-1 viremia had a moderate impact on CD4+ T-cells and HIV-1 viral load, which requires a longitudinal study to identify the beneficial influence on HIV-1 disease progression and outcome.
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Progressão da Doença , Infecções por Flaviviridae , Infecções por HIV , HIV-1 , Carga Viral , Humanos , Índia/epidemiologia , Masculino , Infecções por HIV/virologia , Infecções por HIV/epidemiologia , Infecções por HIV/complicações , Adulto , Feminino , Infecções por Flaviviridae/epidemiologia , Infecções por Flaviviridae/virologia , Prevalência , Pessoa de Meia-Idade , HIV-1/genética , HIV-1/isolamento & purificação , Adulto Jovem , Coinfecção/virologia , Coinfecção/epidemiologia , Genótipo , Contagem de Linfócito CD4 , Pegivirus/genética , Viremia/epidemiologiaRESUMO
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.
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BACKGROUND: Monkeypox (Mpox) is an important human pathogen without etiological treatment. A viral-host interactome study may advance our understanding of molecular pathogenesis and lead to the discovery of suitable therapeutic targets. METHODS: GEO Expression datasets characterizing mRNA profile changes in different host responses to poxviruses were analyzed for shared pathway identification, and then, the Protein-protein interaction (PPI) maps were built. The viral gene expression datasets of Monkeypox virus (MPXV) and Vaccinia virus (VACV) were used to identify the significant viral genes and further investigated for their binding to the library of targeting molecules. RESULTS: Infection with MPXV interferes with various cellular pathways, including interleukin and MAPK signaling. While most host differentially expressed genes (DEGs) are predominantly downregulated upon infection, marked enrichments in histone modifiers and immune-related genes were observed. PPI analysis revealed a set of novel virus-specific protein interactions for the genes in the above functional clusters. The viral DEGs exhibited variable expression patterns in three studied cell types: primary human monocytes, primary human fibroblast, and HeLa, resulting in 118 commonly deregulated proteins. Poxvirus proteins C6R derived protein K7 and K7R of MPXV and VACV were prioritized as targets for potential therapeutic interventions based on their histone-regulating and immunosuppressive properties. In the computational docking and Molecular Dynamics (MD) experiments, these proteins were shown to bind the candidate small molecule S3I-201, which was further prioritized for lead development. RESULTS: MPXV circumvents cellular antiviral defenses by engaging histone modification and immune evasion strategies. C6R-derived protein K7 binding candidate molecule S3I-201 is a priority promising candidate for treating Mpox.
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Interações Hospedeiro-Patógeno , Monkeypox virus , Vaccinia virus , Proteínas Virais , Humanos , Proteínas Virais/genética , Proteínas Virais/metabolismo , Vaccinia virus/genética , Vaccinia virus/metabolismo , Células HeLa , Monkeypox virus/genética , Mpox/virologia , Mapas de Interação de Proteínas , Perfilação da Expressão Gênica , Simulação de Acoplamento Molecular , Poxviridae/genética , Poxviridae/metabolismo , Fibroblastos/virologia , Fibroblastos/metabolismoRESUMO
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.
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Infecções por HIV , HIV-1 , Humanos , HIV-1/genética , Reprodutibilidade dos Testes , Carga Viral/métodos , RNA Viral/genética , Infecções por HIV/diagnóstico , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Human cytomegalovirus (HCMV) reactivation is a major cause of morbidity and mortality among stem cell transplant recipients post-transplantation. AIM: HCMV immediate-early messenger RNA (IE-mRNA) was evaluated as marker of post-transplant HCMV reactivation in bone marrow transplant recipients. METHOD: ology: An in-house real-time reverse transcriptase PCR targeting IE-mRNA was developed to estimate HCMV mRNA levels post-transplantation. Blood samples collected in K2-EDTA tubes from patients (n = 162) admitted with Department of Clinical Hematology were transported in cold condition for routine HCMV DNA screening. For HCMV IE-mRNA quantification, peripheral blood mononuclear cells (PBMCs) were separated from whole blood and stored in RNA later at -70 °C until testing. Samples were collected weekly once for first 3 weeks post-transplantation and thereafter from week 4-12, samples were collected twice weekly. A total of 2467 samples were collected from 162 study participants. RESULTS: Thirty five patients (21.6 %) had post-transplant HCMV reactivation. Twenty five patients with complete follow-up were selected for monitoring HCMV DNA. HCMV IE-mRNA PCR was performed for 15 patients and 7(46.6 %) patients had detectable mRNA levels. HCMV IE-mRNA was detected in all patients with increasing HCMV DNA levels except for one patient in whom IE-mRNA appeared 3 days before HCMV DNA was detected. One patient had detectable HCMV IE-mRNA during declining HCMV DNA level. However the patient showed an increased HCMV DNA one week later, indicating the importance of HCMV mRNA in predicting HCMV replication. CONCLUSION: Quantification of HCMV IE-mRNA may be a valuable tool to predict progression of HCMV infection post-transplantation, with further prospective studies needed for validation.
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Infecções por Citomegalovirus , Citomegalovirus , Humanos , Citomegalovirus/genética , Infecções por Citomegalovirus/diagnóstico , Leucócitos Mononucleares , Estudos Prospectivos , DNA Viral/genética , RNA Mensageiro/genética , Células-Tronco HematopoéticasRESUMO
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.
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Antígenos de Superfície da Hepatite B , Hepatite B , Imunoensaio , Medições Luminescentes , Hepatite B/diagnóstico , Antígenos de Superfície da Hepatite B/análise , Imunoensaio/métodos , Sensibilidade e Especificidade , Humanos , Medições Luminescentes/métodosRESUMO
OBJECTIVE: To determine the accuracy of high-risk human papillomavirus (HPV) DNA samples on filter paper in comparison to specimen transport medium (STM). METHODS: This was a cross-sectional diagnostic study of 42 consecutive women who were prospectively recruited. Each had self-collected vaginal samples on filter paper, physician-collected cervical samples on filter paper, and physician-collected cervical samples in STM. HPV DNA testing was performed with a Hybrid Capture 2 system (Qiagen). Sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV), and agreement of filter paper methods with the standard procedure were calculated. RESULTS: The overall prevalence of HPV in STM was 67.5%. Detection of HPV DNA in the physician-collected cervical samples on filter paper had a sensitivity of 77.8%, a specificity of 100%, a PPV of 100%, and an NPV of 68.4%. The patient's self-sampling on filter paper had a sensitivity of 66.7%, a specificity of 100%, a PPV of 100%, and an NPV of 59.1%. The agreement between STM method and physician-collected sample on filter paper was substantial, (κ = 0.695, P < 0.001), while the agreement between STM and self-collected samples on filter paper was moderate (κ = 0.565, P < 0.001). Most patients reported that self-collection was acceptable (100%), painless (95%), and not embarrassing (95%). CONCLUSION: Filter paper, with dried self-collected vaginal samples, can be used to detect high-risk HPV with acceptable accuracy.
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Infecções por Papillomavirus , Displasia do Colo do Útero , Neoplasias do Colo do Útero , Humanos , Feminino , Papillomavirus Humano , Neoplasias do Colo do Útero/diagnóstico , Infecções por Papillomavirus/diagnóstico , Estudos Transversais , Papillomaviridae/genética , DNA Viral/genética , Manejo de Espécimes/métodos , Esfregaço Vaginal/métodos , Sensibilidade e Especificidade , Detecção Precoce de Câncer/métodos , Displasia do Colo do Útero/diagnósticoRESUMO
It is believed that human papilloma virus infection (HPV), which is caused by the DNA virus, is the most prominent factor contributing to sexually transmitted disease (STD) in the world, with males having a prevalence rate of 3.5%-45% while that women are 2%-44%. Infertility is a rising problem on a global basis, affecting anywhere from 10% to 30% of couples who have reached reproductive age. This study aims to investigate the existing research on HPV, its connection to male infertility, and how it could be a helpful tool for medical professionals managing HPV in the context of reproductive health care. Infection with HPV has been identified as a risk factor for several spontaneous abortions; however, there is a lack of evidence on how HPV influences individuals undergoing assisted reproductive technology (ART) in terms of live births. The significance of the immune response to HPV-infected male reproductive system cells and its effect on embryos, as well as the oxidative stress generated by high-risk HPV DNA damage and genomic instability, is discussed in this review. Further, the association between male individuals infected with HPV and asthenozoospermia should provide a compelling case for vaccinating young people against HPV.
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Infertilidade Masculina , Infecções por Papillomavirus , Gravidez , Humanos , Masculino , Feminino , Adolescente , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/epidemiologia , Papillomavirus Humano , Saúde Reprodutiva , Papillomaviridae/genéticaRESUMO
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.
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Antivirais , Hepatite C Crônica , Humanos , Antivirais/uso terapêutico , Estudos Longitudinais , RNA Viral/genética , Hepacivirus/genética , Antígenos da Hepatite C , Hepatite C Crônica/tratamento farmacológico , Recidiva , GenótipoRESUMO
Epidemiological link between HPV and SLE is evolving. The possibility of HPV infection-induced molecular mimicry and systemic lupus erythematosus (SLE) was elucidated through detailed in silico analyses. Conserved regions in the structural protein sequences of high-risk HPV types were inferred, and sequence homologies between viral and human peptides were identified to delineate proteins implicated in SLE. B-cell epitopes and MHC-class II binding were compiled using Immune Epitope Database and ProPred II analysis tool. Molecular modeling and molecular dynamics/simulation (MDS) were performed using AutoDock Vina and GROMACS, respectively. Sequence alignment revealed 32 conserved regions, and 27/32 viral peptides showed varying similarities to human peptides, rich in B-cell epitopes with superior accessibility, high hydrophilicity, antigenicity and disposition to bind many class-II HLA alleles. Molecular docking of 13 viral peptides homologous (100%) to human peptides implicated in SLE showed that VIR-PEP1 (QLFNKPYWL) and VIR-PEP2 (DTYRFVTS) exhibited higher binding affinities than corresponding human peptides to SLE predisposing HLA-DRB1 allele. MDS of these peptides showed that the viral peptides had superior folding, compactness, and a higher number of hydrogen bonds than human peptides throughout the simulation period. SASA analysis revealed that the VIR-PEP1&2 fluctuated less frequently than corresponding human peptides. MM-PBSA revealed that the VIR-PEP2 complex exhibited higher binding energy than the human peptide complex. This suggests that highly conserved structural peptides of high-risk HPV types homologous to human peptides could compete and bind avidly to the HLA allele associated with SLE and predispose HPV-infected individuals to SLE through molecular mimicry.Communicated by Ramaswamy H. Sarma.
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Lúpus Eritematoso Sistêmico , Infecções por Papillomavirus , Humanos , Epitopos de Linfócito B , Mimetismo Molecular , Simulação de Acoplamento Molecular , Peptídeos/química , Epitopos de Linfócito TRESUMO
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.
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Hepatite A , Antígenos de Superfície da Hepatite B , Hepatite B Crônica , Hepatite B , Humanos , DNA Viral , Hepatite B/diagnóstico , Antígenos de Superfície da Hepatite B/sangue , Antígenos de Superfície da Hepatite B/química , Vírus da Hepatite B , Hepatite B Crônica/diagnóstico , Sensibilidade e EspecificidadeRESUMO
PURPOSE: The COVID-19 pandemic was unique in the history of outbreaks because of the massive scaling up of resources related to diagnostics, treatment modalities, and vaccines. To understand the impact of the pandemic among laboratory professionals, we aimed to conduct a survey to assess the improvement in the lab capacity post-covid in terms of infrastructure and accreditation status across various levels of hospitals and to determine the changes in the practice of infection control precautions during the pandemic. METHODS: This was an anonymous, online-based survey (using 58 item questionnaire) conducted between July 09, 2021, and August 07, 2021. The survey targeted all EQAS registered diagnostic laboratories located in India. RESULTS: The survey reached out to 1182 participants, out of which 721 (61%) laboratories completed the questionnaire. During pre-COVID times, only 39% (282/721) of the laboratories had an RT-PCR facility. Among these 721 labs, 514 used open system RT-PCR assay, 217 labs used Truenat assay, 188 labs used GeneXpert assay, 31 used Abbott ID Now and 350 labs performed rapid antigen tests. During the pandemic, 55.3% got NABL accreditation and 7.4% were in the process of applying for COVID-19 molecular testing. In this, 80.7% of the laboratories participated in the ICMR - COVID quality control assessment. It was estimated that 41.4% of the laboratory professionals were re-using N95 masks. Overall, the infection prevention and control practices varied across each laboratory and hospital. CONCLUSION: These survey findings helped us to understand the strength and efficiency of laboratories in India in setting up new assays during a crisis time. Based on our findings, we propose to connect this network in a sustained manner to efficiently utilize the existing platforms to adapt to future pandemics.
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COVID-19 , Fortalecimento Institucional , Controle de Infecções , Laboratórios Clínicos , Pessoal de Laboratório , Pandemias , Índia/epidemiologia , COVID-19/diagnóstico , COVID-19/epidemiologia , COVID-19/prevenção & controle , Pandemias/prevenção & controle , Garantia da Qualidade dos Cuidados de Saúde , Inquéritos e Questionários , Respiradores N95 , Teste para COVID-19 , Diagnóstico PrecoceRESUMO
Since 2000, a resurgence of syphilis has been noted in many developed and developing countries, especially among men who have sex with men (MSM). Incidence and prevalence of syphilis in pregnant women have been reduced drastically by mandatory screening in early pregnancy. Insufficient data in other populations especially from developing countries limit targeted public health interventions. This study aimed to describe the clinical and epidemiological profile of serologically confirmed syphilis cases among the non-pregnant high-risk group reporting to a tertiary care center in Southern India. A retrospective study was carried out in a tertiary care center in Southern India for 6 years from 2015 to 2020. A total of 265 serologically confirmed syphilis patients were included. A statistically significant increase in positivity from 0.52 to 2.1% was observed in this study (2015 to 2020). Among risk factors, high-risk behavior with multiple heterosexual partners was the commonest (51.3%), followed by marital partners who tested positive (9.4%) and MSM (7.5%). The majority of the patients were diagnosed at the latent stage (79%), followed by secondary syphilis (10%) and tertiary syphilis (8%). A quarter of patients (23%) were coinfected with HIV. Serological non-responsiveness was more common among HIV infected (47 vs. 24%). Sixteen had neurosyphilis and six had ocular involvement. HIV co-infection complicated 50% (8/16) of neurosyphilis patients. Syphilis is still prevalent, especially in high-risk groups including those are attending STI clinics. Further prospective multicentric studies are needed to identify and implement public health measures.
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Infecções por HIV , Neurossífilis , Minorias Sexuais e de Gênero , Sífilis , Adulto , Feminino , Infecções por HIV/epidemiologia , Homossexualidade Masculina , Humanos , Índia/epidemiologia , Masculino , Neurossífilis/complicações , Gravidez , Estudos Retrospectivos , Sífilis/diagnóstico , Sífilis/epidemiologia , Sífilis/prevenção & controle , Centros de Atenção TerciáriaRESUMO
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.
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Infecções por HIV , HIV-2 , Humanos , Carga Viral , HIV-2/genética , Kit de Reagentes para Diagnóstico , Infecções por HIV/diagnóstico , Infecções por HIV/tratamento farmacológico , Reação em Cadeia da Polimerase em Tempo Real , RNA Viral , Sensibilidade e EspecificidadeRESUMO
Gene therapy using an adeno-associated virus (AAV) vector offers a new treatment option for individuals with monogenetic disorders. The major bottleneck is the presence of pre-existing anti-AAV antibodies, which impacts its use. Even very low titers of neutralizing antibodies (NAb) to capsids from natural AAV infections have been reported to inhibit the transduction of intravenously administered AAV in animal models and are associated with limited efficacy in human trials. Assessing the level of pre-existing NAb is important for determining the primary eligibility of patients for AAV vector-based gene therapy clinical trials. Techniques used to screen AAV-antibodies include AAV capsid enzyme-linked immunosorbent assay (ELISA) and transduction inhibition assay (TIA) for detecting total capsid-binding (TAb) and Nab, respectively. In this study, we screened 521 individuals with hemophilia A from India for TAb and NAb using ELISA and TIA, respectively. The prevalence of TAb and NAb in hemophilia A patients from India were 96% and 77.5%, respectively. There was a significant increase in anti-AAV3 NAb prevalence with age in the hemophilia A patient group from India. There was a trend in anti-AAV3 TAb positivity between the pediatric age group (94.4%) and the adult age group (97.4%).
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Anticorpos Antivirais , Hemofilia A , Adulto , Animais , Anticorpos Neutralizantes , Criança , Dependovirus/genética , Vetores Genéticos , Hemofilia A/epidemiologia , Hemofilia A/imunologia , Hemofilia A/terapia , Humanos , Prevalência , SorogrupoRESUMO
Transplantation of allogenic hematopoietic stem and progenitor cells (HSPCs) with C-C chemokine receptor type 5 (CCR5) Δ32 genotype generates HIV-1 resistant immune cells. CCR5 gene edited autologous HSPCs can be a potential alternative to hematopoietic stem cell transplantation (HSCT) from HLA-matched CCR5 null donor. However, the clinical application of gene edited autologous HSPCs is critically limited by the quality of the graft, as HIV also infects the HSPCs. In this study, by using mobilized HSPCs from healthy donors, we show that the CD34+CD90+ hematopoietic stem cells (HSCs) express 7-fold lower CD4/CCR5 HIV receptors, higher levels of SAMHD1 anti-viral restriction factor, and possess lower susceptibility to HIV infection than the CD34+CD90- hematopoietic progenitor cells. Further, the treatment with small molecule cocktail of Resveratrol, UM729 and SR1(RUS) improved the in vivo engraftment potential of CD34+CD90+ HSCs. To demonstrate that CD34+CD90+ HSC population as an ideal graft for HIV gene therapy, we sort purified CD34+CD90+ HSCs, treated with RUS and then gene edited the CCR5 with single sgRNA. On transplantation, 100,000 CD34+CD90+ HSCs were sufficient for long-term repopulation of the entire bone marrow of NBSGW mice. Importantly, the gene editing efficiency of ~90% in the infused product was maintained in vivo, facilitating the generation of CCR5 null immune cells, resistant to HIV infection. Altogether, CCR5 gene editing of CD34+CD90+ HSCs provide an ideal gene manipulation strategy for autologous HSCT based gene therapy for HIV infection.
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Infecções por HIV , Animais , Antígenos CD34/metabolismo , Edição de Genes , Terapia Genética , Células-Tronco Hematopoéticas/metabolismo , CamundongosRESUMO
BACKGROUND: An important aspect of ensuring blood safety is the performance of mandatory serological testing for transfusion transmissible infections. The practice of internal quality control (IQC) in blood banks in India is nonuniform, especially the use of third-party materials. Cited reasons are cost, lack of access to control materials, and need for deep-freezers for storage, if prepared in-house. OBJECTIVE: Validation of dried tube specimen (DTS) from HIV-positive plasma as a low-cost, stable material for use as IQC material in blood banks. METHODS: Fresh-frozen plasma (FFP) prepared from four HIV-positive blood-donors were pooled. Equal numbers of seronegative FFPs were pooled. Twenty microlitre aliquots of plasma were made in micro-centrifuge tubes and air-dried overnight at room-temperature. These were stored in 2-8°C refrigerators and tested once weekly for 6 months on multiple platforms with different detection principles: Rapid tests, second-generation enzyme-linked immunosorbent assay (ELISA), fourth-generation ELISA, and fourth-generation Chemiluminescence immunoassay. The protocol was sustained over the next 6 months with decreased testing frequency to study the extended stability of DTS. RESULTS: A total of 139 positive-DTS and 139 negative-DTS were tested with 100% samples showing consistent results on all platforms over 1 year. There was mild deterioration in reaction strengths, which did not interfere in result interpretations. CONCLUSION: Plasma in form of DTS maintained stability when stored at 2-8°C for 1 year. This provides evidence that DTS can be a modality for the production of cost-effective, stable, in-house control material for resource-restricted countries.
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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.
Assuntos
Coinfecção , Infecções por HIV , HIV-1 , Estudos Transversais , Infecções por HIV/complicações , Vírus da Hepatite B , Humanos , Índia , Carga ViralRESUMO
BACKGROUND: Antiretroviral therapy (ART) has led to a decline in autoimmune diseases but lacks studies on its effect on autoantibodies. METHODS: It is a cross-sectional study with archived samples from 100 paired HIV-1 infected ART naïve and experienced individuals and 100 prospectively collected matched blood-donor controls. Antinuclear antibody, IgG anticardiolipin antibody, IgM and IgG ß2 glycoprotein-1 antibodies, and total IgG levels were detected. Results are expressed as mean with standard deviation (SD), median, percentage positivity, and a p<0.05 is considered significant. The study was approved by the Institutional Review Board. RESULTS: The median viral load of the treatment naïve samples was 4.34 Log copies/mL, while all were virally suppressed post ART with a median duration of treatment for 12 months (range: 3-36 months). The percentage of antinuclear antibody positivity was 5% among ART naïve and controls, with a decrease of 2% post ART (p= 0.441). The positivity for anti-cardiolipin antibody was 15% among ART naïve while none of the ART experienced or controls were positive (p<0.05). IgM ß2 glycoprotein-1 were 4%, 1% and 3% among ART naïve, treated and controls, respectively (p<0.05). IgG ß2 glycoprotein-1 was 2% among ART naïve while none of the treated and controls were positive (p<0.05). The mean total IgG level among ART naïve, experienced, and controls were 21.82 (SD 6.67), 16.91 (SD 3.38), 13.70 (SD 2.24) grams/Litre, respectively (p<0.05). CONCLUSION: ART has a significant effect on IgG anti-cardiolipin antibody and total IgG but only a marginal effect on ANA, IgM, and IgG ß2 glycoprotein-1 antibodies.
Assuntos
Fármacos Anti-HIV/imunologia , Fármacos Anti-HIV/uso terapêutico , Autoanticorpos/sangue , Autoanticorpos/efeitos dos fármacos , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Carga Viral/efeitos dos fármacos , Adulto , Anticorpos Anticardiolipina/sangue , Estudos Transversais , Humanos , Imunoglobulina G/sangue , Pessoa de Meia-Idade , beta 2-Glicoproteína I/sangueRESUMO
Adeno-associated virus (AAV) vector-based gene therapy offers a new treatment option for individuals with hemophilia. Pre-existing anti-AAV antibodies significantly impact the use of AAV vectors. Even relatively low titers of AAV neutralizing antibodies (NAb) from natural AAV infections against the capsid have been shown to inhibit the transduction of intravenously administered AAV in animal models and were associated with limited efficacy in human trials. This is important for determining the primary eligibility of patients for AAV vector-based gene therapy clinical trials. Current techniques to screen AAV antibodies include AAV capsid enzyme-linked immunosorbent assay (ELISA) for total antibodies and a transduction inhibition assay (TIA) for NAb. This study developed and screened total capsid binding anti-AAV3 antibodies by using ELISA and determined NAb levels by TIA using mCherry flow cytometry in healthy individuals with hemophilia B in India. One hundred and forty-three apparently healthy controls and 92 individuals with hemophilia B were screened. The prevalence of total and NAb in healthy controls was 79.7% and 65%, respectively; the prevalence of total and NAb in patients with hemophilia B for AAV3 was 92.4% and 91.3%, respectively.