A Recurrent Nonsense Mutation in NECTIN4 Underlying Ectodermal Dysplasia-Syndactyly Syndrome with a Novel Phenotype in a Consanguineous Kashmiri Family.

EDSS1, a syndrome characterized by ectodermal dysplasia-syndactyly, is inherited in an autosomal recessive manner due to mutations in the NECTIN4/PVRL4 gene. Clinical manifestations of the syndrome include defective nail plate, sparse to absent scalp and body hair, spaced teeth with enamel hypoplasia, and bilateral cutaneous syndactyly in the fingers and toes. Here, we report a consanguineous family of Kashmiri origin presenting features of EDSS1. Using whole exome sequencing, we found a recurrent nonsense mutation (NM_030916: c.181C > T, p.(Gln61 ∗)) in the NECTIN4 gene. The variant segregated perfectly with the disorder within the family. The candidate variant was absent in 50 in-house exomes pertaining to other disorders from the same population. In addition to the previously reported clinical phenotype, an upper lip cleft was found in one of the affected members as a novel phenotype that is not reported by previous studies in EDSS1 patients. Therefore, the study presented here, which was conducted on the Kashmiri population, is the first to document a NECTIN4 mutation associated with the upper lip cleft as a novel phenotype. This finding broadens the molecular and phenotypic spectrum of EDSS1.

Designing a multi-epitopes subunit vaccine against human herpes virus 6A based on molecular dynamics and immune stimulation.

Human Herpesvirus 6A (HHV-6A) is a prevalent virus associated with various clinical manifestations, including neurological disorders, autoimmune diseases, and promotes tumor cell growth. HHV-6A is an enveloped, double-stranded DNA virus with a genome of approximately 160-170 kb containing a hundred open-reading frames. An immunoinformatics approach was applied to predict high immunogenic and non-allergenic CTL, HTL, and B cell epitopes and design a multi-epitope subunit vaccine based on HHV-6A glycoprotein B (gB), glycoprotein H (gH), and glycoprotein Q (gQ). The stability and correct folding of the modeled vaccines were confirmed through molecular dynamics simulation. Molecular docking found that the designed vaccines have a strong binding network with human TLR3, with Kd values of 1.5E-11 mol/L, 2.6E-12 mol/L, 6.5E-13 mol/L, and 7.1E-11 mol/L for gB-TLR3, gH-TLR3, gQ-TLR3, and the combined vaccine-TLR3, respectively. The codon adaptation index values of the vaccines were above 0.8, and their GC content was around 67 % (normal range 30-70 %), indicating their potential for high expression. Immune simulation analysis demonstrated robust immune responses against the vaccine, with approximately 650,000/ml combined IgG and IgM antibody titer. This study lays a strong foundation for developing a safe and effective vaccine against HHV-6A, with significant implications for treating associated conditions.

Core amino acid substitutions in HCV-3a isolates from Pakistan and opportunities for multi-epitopic vaccines.

Hepatitis C virus (HCV), which infected 71 million worldwide and about 5%-6% are from Pakistan, is an ssRNA virus, responsible for end-stage liver disease. To date, no effective therapy is available to cure this disease. Hence, it is important to study the most prevalent genotypes infecting human population and design novel vaccine or small molecule inhibitors to control the infections associated with HCV. Therefore, in this study clinical samples (n = 35; HCV-3a) from HCV patients were subjected to Sanger sequencing method. The sequencing of the core gene, which is generally considered as conserved, involved in the detection, quantitation and genotyping of HCV was performed. Multiple mutations, that is, R46C, R70Q, L91C, G60E, N/S105A, P108A, N110I, S116V, G90S, A77G and G145R that could be linked with response to antiviral therapies were detected. Phylogenetic analysis suggests emerging viral isolates are circulating in Pakistan. Using ab initio modelling technique, we predicted the 3D structure of core protein and subjected to molecular dynamics simulation to extract the most stable conformation of the structure for further analysis. Immunoinformatic approaches were used to propose a multi-epitopes vaccine against HCV by using core protein. The vaccine constructs consist of nine CTL and three HTL epitopes joined by different linkers were docked against the two reported Toll-like receptors (TLR-3 and TLR-8). Docking of vaccine construct with TLR-3 and TLR-8 shows proper binding and in silico expression of the vaccine resulted in a CAI value of 0.93. These analyses suggest that specific immune responses may be produced by the proposed vaccine.Communicated by Ramaswamy H. Sarma.

Proteome-wide mapping and reverse vaccinology-based B and T cell multi-epitope subunit vaccine designing for immune response reinforcement against Porphyromonas gingivalis.

Porphyromonas gingivalis, a prominent pathogen responsible for acute periodontal diseases, is widely studied by the scientific community for its successful evasion of the host immune system. P. gingivalis is associated with rheumatoid arthritis, dementia, and Alzheimer's. The pathogen successfully survives itself against the heavy load of conventional antibiotics because of its ability to evade the host immune system. Subtractive proteomics and reverse vaccinology approaches were employed in order to prioritize the best proteins for vaccine designing. Three vaccine candidates with Uniprot ID: Q7MWZ2 (histidine Kinase), Q7MVL1 (Fe (2+) transporter), and Q7MWZ2 (Capsular polysaccharide transport protein) were identified for vaccine designing. These proteins are antigenic and essential for pathogen survival. A wide range of immunoinformatics tools was applied for the prediction of epitopes, B, and T cells, for the vaccine candidate proteins. Molecular docking of the predicted epitopes against the MHC molecules were carried out. In-silico vaccine was constructed using carefully evaluated epitopes and consequently modeled for docking with human Toll-like receptor 2. Chain C of Pam3CSK4 (PDB ID; 2Z7X) was linked to the vaccine as an adjuvant to boost immune response towards the vaccine. For stability evaluation of the vaccine-TLR-2 docked complex, Molecular Dynamics simulations were performed. The reverse-translated nucleotide sequence cloned in Eschericia coli to attain the maximal expression of the vaccine protein. The maximal expression was ensured by CAI score of 0.96. The current vaccine requires future experimental validation to confirm its effectiveness. The vaccine developed will be helpful to protect against P. gingivalis associated infections.Communicated by Ramaswamy H. Sarma.

Abrogation of SARS-CoV-2 interaction with host (NRP1) neuropilin-1 receptor through high-affinity marine natural compounds to curtail the infectivity: A structural-dynamics data.

The evolution of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants around the globe has made the coronavirus disease 2019 (COVID-19) pandemic more worrisome, pressuring the health care system and resulting in an increased mortality rate. Recent studies recognized neuropilin-1 (NRP1) as a key facilitator in the invasion of the new SARS-CoV-2 into the host cell. Therefore, it is considered an imperative drug target for the treatment of COVID-19. Hence, a thorough analysis was needed to understand the impact and to guide new therapeutics development. In this study, we used structural and biomolecular simulation techniques to identify novel marine natural products which could block this receptor and stop the virus entry. We discovered that the binding affinity of CMNPD10175, CMNPD10017, CMNPD10114, CMNPD10115, CMNPD10020. CMNPD10018, CMNPD10153, CMNPD10149 CMNPD10464 and CMNPD10019 were substantial during the virtual screening (VS). We further explored these compounds by analyzing their absorption, distribution, metabolism, and excretion and toxicity (ADMET) properties and structural-dynamics features. Free energy calculations further established that all the compounds exhibit stronger binding energy for NRP1. Consequently, we hypothesized that these compounds might be the best lead candidates for therapeutic interventions hindering virus binding to the host cell. This study provides a strong impetus to develop novel drugs against the SARS-CoV-2 by targeting NRP1.

Effect of temperature and humidity on coronavirus infection in Pakistan.

Ongoing Coronavirus epidemic (COVID-19) identified first in Wuhan, China posed huge impact on public health and economy around the globe. Both cough and sneeze based droplets or aerosols encapsulated COVID-19 particles are responsible for airborne transmission of this virus and caused an unexpected escalation and high mortality worldwide. Current study intends to investigate the correlation of COVID-19 epidemic with meteorological parameters, particularly temperature and humidity. A data set of Epidemiological data of COVID-19 for highly infected provinces of Pakistan was collected from the official website of (https://www.covid.gov.pk/) and weather data was collected from (https://www.timeanddate.com/) during the time period of 1st March to 30th September 2020. The GrapPad prism 5 Software was used to calculate the mean and standard error of mean (SEM). In the current study the incident of daily covid cases is recorded higher in the month of June while the less number of case were reported in the month of May as compared to the other months (April, May, June, July, September and August) in the four province of Pakistan. We also find out that the incident of Covid19 were high at higher temperature (like the average temperature in the month of June 37 °C) while less cases were reported in May the average temperature was 29.5 °C. Furthermore the incident of covid cases were less reported at low humidity while more intendant with high humidity. Pearson's (r) determine the strength of the relationship between the variables. Pearson's correlation coefficient test employed for data analysis revealed that temperature average (TA) and average humidity is not a significant correlated with COVID-19 pandemic. The results obtained from the current analysis for selected parameters indirect correlation of COVID-19 transmission with temperature variation, and humidity. In the present study association of parameters is not correlated with COVID-19 pandemic, suggested need of more strict actions and control measures for highly populated cities. These findings will be helpful for health regulatory authorities and policy makers to take specific measures to combat COVID-19 epidemic in Pakistan.

Mutational analysis of the spike protein of SARS-COV-2 isolates revealed atomistic features responsible for higher binding and infectivity.

Introduction: The perpetual appearance of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-COV-2), and its new variants devastated the public health and social fabric around the world. Understanding the genomic patterns and connecting them to phenotypic attributes is of great interest to devise a treatment strategy to control this pandemic. Materials and Methods: In this regard, computational methods to understand the evolution, dynamics and mutational spectrum of SARS-CoV-2 and its new variants are significantly important. Thus, herein, we used computational methods to screen the genomes of SARS-CoV-2 isolated from Pakistan and connect them to the phenotypic attributes of spike protein; we used stability-function correlation methods, protein-protein docking, and molecular dynamics simulation. Results: Using the Global initiative on sharing all influenza data (GISAID) a total of 21 unique mutations were identified, among which five were reported as stabilizing while 16 were destabilizing revealed through mCSM, DynaMut 2.0, and I-Mutant servers. Protein-protein docking with Angiotensin-converting enzyme 2 (ACE2) and monoclonal antibody (4A8) revealed that mutation G446V in the receptor-binding domain; R102S and G181V in the N-terminal domain (NTD) significantly affected the binding and thus increased the infectivity. The interaction pattern also revealed significant variations in the hydrogen bonding, salt bridges and non-bonded contact networks. The structural-dynamic features of these mutations revealed the global dynamic trend and the finding energy calculation further established that the G446V mutation increases the binding affinity towards ACE2 while R102S and G181V help in evading the host immune response. The other mutations reported supplement these processes indirectly. The binding free energy results revealed that wild type-RBD has a TBE of -60.55 kcal/mol while G446V-RBD reported a TBE of -73.49 kcal/mol. On the other hand, wild type-NTD reported -67.77 kcal/mol of TBE, R102S-NTD reported -51.25 kcal/mol of TBE while G181V-NTD reported a TBE of -63.68 kcal/mol. Conclusions: In conclusion, the current findings revealed basis for higher infectivity and immune evasion associated with the aforementioned mutations and structure-based drug discovery against such variants.

Guillain-Barre syndrome: An autoimmune disorder post-COVID-19 vaccination?

SARS-CoV-2 causes Coronavirus Disease 2019 (COVID-19), an infectious condition that can present none or one or more of these symptoms: fever, cough, headache, sore throat, loss of taste and smell, aches, fatigue and musculoskeletal pain. For the prevention of COVID-19, there are vaccines available including those developed by Pfizer, Moderna, Sinovac, Janssen, and AstraZeneca. Recent evidence has shown that some COVID-19-vaccinated individuals can occasionally develop as a potential side effect Guillain-Barre syndrome (GBS), a severe neurological autoimmune condition in which the immune response against the peripheral nerve system (PNS) can result in significant morbidity. GBS had been linked previously to several viral or bacterial infections, and the finding of GBS after vaccination with certain COVID-19, while rare, should alert medical practitioners for an early diagnosis and targeted treatment. Here we review five cases of GBS that developed in different countries after COVID-19 vaccination.

Hepatitis C virus infection in garbage pickers of different districts of Khyber Pakhtunkhwa, Pakistan.

Hepatitis C Virus (HCV) is a significant health problem worldwide, especially in developing countries. Medical municipal waste pickers are at higher risk of exposure to infectious viral diseases. The current study aimed to explore HCV infection in different waste collectors. The objective of the current study was to investigate the incidence of HCV infection and associated risks among the waste collectors of five districts of Malakand Divisions and Bajaur district (old Bajaur agency) Khyber Pakhtunkhwa, Pakistan. During the study period (May-December 2017), blood samples were collected from 300 waste handlers and tested for anti-HCV antibodies using the ICT method. The results obtained from the data were analyzed statistically using SPSS 20 version. In this study, we included 300 males aged 9 to 76 years, which means 27.31 ± 11.9 years. The prevalence of HCV was 3.3% (10/300). Among the positive cases, HCV infection was slightly higher in married persons than in unmarried (3% vs. 0.33%, %, PV < 0.01, odds ratio 4.73, 95% CI =2.2-9.8). A high infection rate was reported in waste scavengers from the district Swat, followed by those in the Bajaur district. Needle prick injuries and barehanded practices were the significant risk factors for infected cases. Our findings highlight the need for personal protective equipment. It is suggested that all garbage collectors should be trained in handling waste to reduce infection.

Incidence of malarial infection and response to antimalarial drugs at Districts Lower Dir and Swat of Khyber Pakhtunkhwa, Pakistan.

Malaria is the leading cause of mortality and morbidity all over the world. Several antimalarial drugs are available for the treatment of malaria. The main objective of this study was to investigate the incidence of malarial infection and the use of prescribed antimalarial drugs. A cross-sectional study was carried out to collect quantitative data from selected sites in District Lower Dir and Swat of Malakand Division Khyber Pakhtunkhwa (K.P.), Pakistan. Screening of selected patients was performed using both thick and thin films and was observed with the help of a light microscope. In this study, a total of 2517 blood samples were tested. Overall positive infection was 12% Plasmodium vivax (99.07%) and Plasmodium falciparum (0.92%). Our results evaluate that infection with Plasmodium vivax was higher than Plasmodium falciparum. No other Plasmodium species or mixed infections were observed. The rate of infection was more frequent in males as compared to female patients. The highest percentage was recorded in the summer season (35.07%), while the lowest was documented in the winter (11.7%). Out of 325 patients, 311 (95.7%) were treated with Chloroquine, and the remaining were treated with Artemether. Chloroquine was used as a drug of choice for Plasmodium vivax infection. The present study concludes that Plasmodium vivax and Plasmodium falciparum are the two common agents for malaria in Malakand Division. However, Plasmodium vivax was dominant over Plasmodium falciparum. The infection rate was high in males from District Lower Dir during the summer season.

Annotation of Potential Vaccine Targets and Design of a Multi-Epitope Subunit Vaccine against Yersinia pestis through Reverse Vaccinology and Validation through an Agent-Based Modeling Approach.

Yersinia pestis is responsible for plague and major pandemics in Asia and Europe. This bacterium has shown resistance to an array of drugs commonly used for the treatment of plague. Therefore, effective therapeutics measurements, such as designing a vaccine that can effectively and safely prevent Y. pestis infection, are of high interest. To fast-track vaccine development against Yersinia pestis, herein, proteome-wide vaccine target annotation was performed, and structural vaccinology-assisted epitopes were predicted. Among the total 3909 proteins, only 5 (rstB, YPO2385, hmuR, flaA1a, and psaB) were shortlisted as essential vaccine targets. These targets were then subjected to multi-epitope vaccine design using different linkers. EAAK, AAY, and GPGPG as linkers were used to link CTL, HTL, and B-cell epitopes, and an adjuvant (beta defensin) was also added at the N-terminal of the MEVC. Physiochemical characterization, such as determination of the instability index, theoretical pI, half-life, aliphatic index, stability profiling, antigenicity, allergenicity, and hydropathy of the ensemble, showed that the vaccine is highly stable, antigenic, and non-allergenic and produces multiple interactions with immune receptors upon docking. In addition, molecular dynamics simulation confirmed the stable binding and good dynamic properties of the vaccine-TLR complex. Furthermore, in silico and immune simulation of the developed MEVC for Y. pestis showed that the vaccine triggered strong immune response after several doses at different intervals. Neutralization of the antigen was observed at the third day of injection. Conclusively, the vaccine designed here for Y. pestis produces an immune response; however, further immunological testing is needed to unveil its real efficacy.

Exploring the therapeutic potential of Hibiscus rosa sinensis synthesized cobalt oxide (Co3O4-NPs) and magnesium oxide nanoparticles (MgO-NPs).

Herein, we present a green, economic and ecofriendly protocol for synthesis of cobalt oxide (Co3O4-NPs) and magnesium oxide nanoparticles (MgO-NPs) for multifaceted biomedical applications. In the study, a simple aqueous leaf extract of Hibiscus rosa sinensis, was employed for the facile one pot synthesis of Co3O4-NPs and MgO-NPs. The well characterized NPs were explored for multiple biomedical applications including bactericidal activity against urinary tract infection (UTI) isolates, leishmaniasis, larvicidal, antidiabetic antioxidant and biocompatibility studies. Our results showed that both the NPs were highly active against multidrug resistant UTI isolates as compared to traditional antibiotics and induced significant zone of inhibition against Proteus Vulgaris, Pseudomonas Aurigenosa and E.coli. The NPs, in particular Co3O4-NPs also showed significant larvicidal activity against the Aedes Aegypti, the mosquitoes involve in the transmission of Dengue fever. Similarly, excellent leishmanicidal activity was also observed against both the promastigote and amastigote forms of the parasite. Furthermore, the particles also exhibited considerable antidiabetic activity by inhibiting α-amylase and α-glucosidase enzymes. The biosynthesized NPs were found to be excellent antioxidant and biocompatible nanomaterials. Owing to ecofriendly synthesis, non-toxic and biocompatible nature, the Hibiscus rosa sinensis synthesized Co3O4-NPs and MgO-NPs can be exploited as potential candidates for multiple biomedical applications.

In Silico Mutagenesis-Based Remodelling of SARS-CoV-1 Peptide (ATLQAIAS) to Inhibit SARS-CoV-2: Structural-Dynamics and Free Energy Calculations.

The prolific spread of COVID-19 caused by a novel coronavirus (SARS-CoV-2) from its epicenter in Wuhan, China, to every nook and cranny of the world after December 2019, jeopardize the prevailing health system in the world and has raised serious concerns about human safety. Multi-directional efforts are made to design small molecule inhibitors, and vaccines and many other therapeutic options are practiced, but their final therapeutic potential is still to be tested. Using the old drug or vaccine or peptides could aid this process to avoid such long experimental procedures. Hence, here, we have repurposed a small peptide (ATLQAIAS) from the previous study, which reported the inhibitory effects of this peptide. We used in silico mutagenesis approach to design more peptides from the native wild peptide, which revealed that substitutions (T2W, T2Y, L3R, and A5W) could increase the binding affinity of the peptide towards the 3CLpro. Furthermore, using MD simulation and free energy calculation confirmed its dynamics stability and stronger binding affinities. Per-residue energy decomposition analysis revealed that the specified substitution significantly increased the binding affinity at the residue level. Our wide-ranging analyses of binding affinities disclosed that our designed peptide owns the potential to hinder the SARS-CoV-2 and will reduce the progression of SARS-CoV-2-borne pneumonia. Our research strongly suggests the experimental and clinical validation of these peptides to curtail the recent corona outbreak.

A Correlation among the COVID-19 Spread, Particulate Matters, and Angiotensin-Converting Enzyme 2: A Review.

Air pollution (AP) is one of the leading causes of health risks because it causes widespread morbidity and mortality every year. Its impact on the environment includes acid rain and decreased visibility, but more importantly, it also has an impact on human health. The rise of COVID-19 demonstrates the cost of failing to manage AP. COVID-19 can be spread through the air, and atmospheric particulate matters (PMs) can create a good atmosphere for the long-distance spread of the virus. Moreover, these PMs can cause lung cell inflammation, thereby increasing sensitivity and the severity of symptoms in COVID-19 patients. In this study, we emphasized the potential role of PMs in the spread of COVID-19. The relationship among COVID-19, PMs, and angiotensin-converting enzyme 2 (ACE2) (receptor involved in virus entry into lung cells and inflammation) was also summarized.

Excessive use of disinfectants against COVID-19 posing a potential threat to living beings.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan city of China in late December 2019 and identified as a novel coronavirus disease 2019 (COVID-19). On January 30, the World Health Organization (WHO) declared the coronavirus outbreak a global public health emergency. The rapid spread of the pathogen across the communities shock the entire population. As no existing therapy were available during the pandemic. Health professionals recommended frequent washing of hands with soap and alcohol-based sanitizers. Disinfectants were extensively sprayed to minimize the possibility of getting COVID-19. Despite the potential benefits of these germicidal agents against COVID-19. Alcohol-based hand sanitizers lead to dry skin, infection, and alcohol poisoning. Children are considered more prone to alcohol poisoning and other major health concern. Precautionary measures should be ensured to protect the community from the possible risk associated with disinfectants.

Vitamin D exerts neuroprotection via SIRT1/nrf-2/ NF-kB signaling pathways against D-galactose-induced memory impairment in adult mice.

Vitamin D (Vt. D) is one of the vital hormone having multiple functions in various tissues, including brain. Several evidences reported that Vt. D plays a significant part in memory and cognition as its inadequate amount may accelerate cognitive impairment. This study shows for the first time the antioxidant potential of Vt. D against D-Galactose (D-gal) induced oxidative stress mediated Alzheimer disease (AD) pathology in male adult albino mice. The result reveals that the mice exposed to D-gal (120 mg/kg) for eight weeks have pre-and post-synaptic dysfunction and impaired memory investigated through Morris water maze and Y-maze tests. This is followed by the suppressed Nuclear factor erythroid 2-related factor 2 (NRF2), Heme Oxygenase-1 (HO-1) and elevated expressions of Nuclear Factor kappa B (NF-kB), Tumor Necrosis Factor alpha (TNF-α) and Interleukin 1 beta (IL-1ß) proteins in the brain homogenates evaluated through western blotting technique. On the other hand Vt. D (100 µg/kg) administration (three times a week for 4 weeks) activated Silent mating type information regulation 2 homolog 1 (SIRT1) and significantly improved both the neuronal synapse and memory, reduced oxidative stress by upregulating NRF-2 and HO-1 and downregulating NF-kB, TNF-α and IL-1ß proteins expression. Most importantly, Vt. D significantly abrogate the amyloidogenic pathway of amyloid beta (Aß) production against D-gal in the brains of adult male albino mice. These results reveal that Vt. D being an antioxidant agent plays a vital role in reducing the AD pathophysiology in D-gal induced animal model of aging, therefore act as a potential drug candidate in neurodegenerative diseases.

The Absence of HCV RNA and NS5A Protein in Peripheral Blood Mononuclear Cells Is a Prognostic Tool for Sustained Virological Response.

Hepatitis C Virus (HCV) infection is a major health concern worldwide. The presence of both HCV viral RNA and NS5A proteins in peripheral blood mononuclear cells (PBMCs) indicate the efficacy of the treatment during sustained virological response (SVR) and end of treatment response (ETR). The main objective of this study was to detect the absence or presence of HCV RNA and NS5A proteins in PBMCs. Blood samples were taken from selected patients (Islamabad, Pakistan) before treatment, at ETR, and during SVR. Two hundred HCV responders to pegylated IFN-α-2a plus ribavirin were selected. HCV RNA was extracted from the patients to determine the viral load by reverse transcription (RT)-polymerase chain reaction before treatment. Out of 200 patients, 152 (76%) and 48 (24%) achieved positive and negative ETR, respectively. Among ETR patients, 134 (88.2%) showed SVR, whereas 18 (11.8%) displayed relapse. The male to female ratio was 92:108 with mean age of 37.4 years. Among 152 ETR-positive patients, 29 (19%) patients' PBMCs were positive for HCV RNA and 27 (17.8%) were positive for NS55A proteins. Patients having HCV RNA in PBMCs showed higher relapse frequency compared with patients lacking it. Similarly, patients having NS5A protein showed significantly higher relapse frequency compared with patients lacking it. All PBMC-positive samples were of genotype 3a. In addition, patients with positive NS5A in their PBMCs showed greater risk of relapse compared with patients having HCV RNA. We conclude that the absence of both viral HCV and proteins can be used as an indicator for diagnosis of SVR in the future.

Distribution and clinical significance of hepatitis B virus genotypes in Pakistan.

BACKGROUND: Hepatitis B virus (HBV) genotype and its role in disease progression and patients' response to antiviral treatment, is not well studied in Pakistan. This comprehensive study was aimed to determine the distribution of HBV genotypes in Pakistan and their possible association with phases of HBV infection. METHODS: A total of 840 HBsAg positive samples was collected and tested for HBV DNA quantity. Samples below 100 IU/ml were excluded from the study. A total of 715 samples representing all the six parts of the country were genotyped by type specific primer PCR method. Clinical data of only 384 patients was compared as the remaining 332 were either receiving antiviral treatment or their infection phase was not confirmed. RESULTS: Genotype D was found in 509 samples (71.2 %), genotype A in 55 samples (7.7 %) and mixed infection with genotypes A and D in 124 samples (17.3 %). Genotypes B, C and E were identified in less than 1 % of the total samples. Genotype A, D and their mixture (A + D) were compared for severity of HBV infection. Significant differences were not found in distribution of HBV genotypes among different disease stages. CONCLUSION: HBV genotype D was the predominant infection in all study areas of Pakistan followed by mixed genotypes infection (A + D) whereas genotype A has 10 times lower prevalence than genotype D. Genotypes B, C, E and F altogether make only 1.5 % of the prevalence. Genotype do not appears to show the severity of liver disease.

Presence of HCV RNA in peripheral blood mononuclear cells may predict patients response to interferon and ribavirin therapy.

BACKGROUND AND OBJECTIVES: Hepatitis C virus (HCV) is considered a hepatotropic virus, but it can repli.cate in peripheral blood mononuclear cells (PBMCs), which influence the sustained virological response (SVR) of the patients, as well as relapse in successfully treated patients. The main objective of this study was to establish the importance of PBMC HCV RNA detection as a primary test to declare the patient as a responder, and the secondary objective was to investigate the risk of non-SVR or relapse in individuals who showed an end-of-treatment (ETR). DESIGN AND SETTINGS: Blood samples were collected after the completion of 6 months of therapy, and they were collected 6 months after the completion of treatment. PATIENTS AND METHODS: A total 103 patients infected with the 3a genotype of HCV and those who were treated with interferon-a-2b and ribavirin for 24 weeks were selected. HCV RNA in plasma of at the end of treatment and 6 months after the completion of treatment was determined with the help of quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: Of the 103 patients, 74.8% (number [n]=77) were end-of-treatment responders, while 25.2% (n=26) were nonresponders. Seventy-seven responders were tested for HCV RNA in their PBMCs. The HCV RNA was detected in the PBMCs of 29 patients (37.7%). After 6 months of the end of treatment, 15 (19.5%) of 77 ETR patients showed virological relapse, while 62 (80.5%) patients attained SVR. Relapse appeared significantly more often in patients with HCV RNA in their PBMCs at the ETR stage when compared to the patients who did not have the viral RNA (34.5% versus 10.4%, respectively; R2=6.67, P=.01; odds ratio [OR]: 1.3; 95% confidence interval [CI]=1.032-1.811). CONCLUSION: Patients with HCV RNA in their PBMCs after attaining an ETR are more likely to show relapse as compared to patients who are negative for viral RNA in PBMCs at the ETR stage.