Prevalence of HPV types in HIV-positive and negative females with normal cervical cytology or dysplasia.

The burden of HPV varies by country and HIV status. The study aimed to evaluate HPV types prevalent in HIV-positive females compared with HIV-negative females in the local population of the federal capital territory in Pakistan. METHOD: The selected female population consisted of 65 already diagnosed HIV-positive females and 135 HIV-negative females. Cervical scrap was collected and analyzed for HPV and cytology. RESULTS: The prevalence of HPV in HIV-positive patients was 36.9%, higher than HIV-negative patients (4.4%). 12.30% had cervical cytology interpreted as "LSIL" and 87.69% had cytology interpreted as "NIL." The high-risk type was detected in 15.39% while 21.54% showed low-risk HPV types. Among the high-risk types, HPV18 (6.15%), HPV16 (4.62%), HPV45 (3.07%), HPV33 (1.53%), HPV58 (3.07%), and HPV68 (1.53%) were found. In patients with LSIL, high-risk HPV accounts for 62.5%. Risk factors, such as age, marital status, educational status, residence, parity, other STDs, and contraceptives, were analyzed to find the correlation with HPV infection Age ≤35 years (OR 1.21, 95% CI, 0.44-3.34), illiterate and incomplete secondary education (OR 1.08, 95% CI, 0.37-3.15), and those reported not to use contraceptives (OR: 1.90; 95% CI: 0.67-5.42) have an association for increased risk of HPV infection. CONCLUSION: HPV18, HPV16, HPV58, HPV45, HPV68, and HPV33 were identified among high-risk HPV types. High-risk HPV was detected in 62.5% of low-grade squamous intraepithelial lesions. The data is useful for health policymakers to develop a strategy for HPV screening and prophylactic vaccination to prevent cervical cancer.

Antibody Response to SARS-CoV-2 in Relation to the Contributing Factors in COVID-19 Patients.

Human health has always been challenged by variety of viral infections, but severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has surpassed all previous viral diseases and emerged as a major health challenge around the globe. Real-time polymerase chain reaction (PCR) is the gold standard for the diagnosis of SARS-CoV-2 and serological assay provides a compliment to diagnosis after second week of infection. The aim of the study is the characterization of antibody response to SARS-CoV-2 in the blood sample of diagnosed coronavirus disease 2019 (COVID-19) patients, and its potential association with factors such as age, gender, time, and symptoms. Serum from 248 confirmed SARS-CoV-2 patients was investigated for antibodies. Elecsys anti-SARS chemiluminescent immune assay was performed for the detection of nucleocapsid-specific antibodies. Association of antibody response with gender, age, and time after onset of symptoms was analyzed. Among 248 PCR positive SARS-CoV-2 patients, 214 (86.3%) have virus-specific antibody signals. Antibodies positivity rate was higher in male patient patients as compared with female patients (90.8% vs. 79.2%, p = 0.009). Patients aged 30-40 years had the highest antibody positivity rate as compared with other groups (89.10%, p = 0.04). Patients age group >60 years had a lower positivity rate (75%, p = 0.04). The increasing trend in the antibodies detection with time was observed, maximum positive antibodies response rate observed at 8 weeks. Patients were categorized on the basis of clinical symptoms into asymptomatic, mild, and moderate; 17.7% were asymptomatic, 60.5% showed mild symptoms, and 21.8% showed moderate symptoms of the disease. Males were seen to be more asymptomatic as compared with females (i.e., 59.1% to 40.9%). The serological test for SARS-CoV-2 has a high sensitivity at >2 weeks after the positive PCR result or onset of illness. In addition, the serological response differs among patients based on gender, age, as well as time between the onset of symptoms or PCR confirmation and sample collection for the study of antibody response.

Designing a multi-epitope vaccine against Mycobacteroides abscessus by pangenome-reverse vaccinology.

Mycobacteroides abscessus (Previously Mycobacterium abscessus) is an emerging microorganism of the newly defined genera Mycobacteroides that causes mainly skin and tissue diseases in humans. The recent availability of total 34 fully sequenced genomes of different strains belonging to this species has provided an opportunity to utilize this genomics data to gain novel insights and guide the development of specific antimicrobial therapies. In the present study, we collected collectively 34 complete genome sequences of M. abscessus from the NCBI GenBank database. Pangenome analysis was conducted on these genomes to understand the genetic diversity and to obtain proteins associated with its core genome. These core proteins were then subjected to various subtractive filters to identify potential antigenic targets that were subjected to multi-epitope vaccine design. Our analysis projected the open pangenome of M. abscessus containing 3443 core genes. After applying various stepwise filtration steps on the core proteins, a total of four potential antigenic targets were identified. Utilizing their constituent CD4 and CD8 T-cell epitopes, a multi-epitope based subunit vaccine was computationally designed. Sequence-based analysis as well as structural characterization revealed the immunological effectiveness of this designed vaccine. Further molecular docking, molecular dynamics simulation and binding free energy estimation with Toll-like receptor 2 indicated strong structural associations of the vaccine with the immune receptor. The promising results are encouraging and need to be validated by additional wet laboratory studies for confirmation.

Indeterminate Prediction of Hepatitis C Virus Genotype by Commercial Real-Time Polymerase Chain Reaction Assay Resolving by Sequencing to Avoid the Consequence of Inaccurate Typing.

Pakistan is ranked second highest after Egypt in hepatitis C virus (HCV) infection. Accurate typing is mandatory to be compliant with the World Health Organization strategy to eliminate HCV infection in 2030. We characterized the HCV genotypes using Abbott real-time polymerase chain reaction assay and indeterminate samples were sequenced. We also investigated the distribution of HCV genotype among different age groups and gender in chronic HCV patients. One thousand thirteen samples were tested for HCV genotyping using Abbott real-time HCV genotyping assay. RNA extraction from plasma was done using the m2000sp platform. The amplification and detection of genotypes was done on m2000rt instrument. The lower limit of detection assay is 500 IU/mL. The indeterminate genotypes were analyzed by sequencing of the NS5B region. We found genotype 1 in 1.68%, genotype 1b in 0.89%, genotype 1a in 0.79%, genotype 2 in 0.6, genotype 3 in 94.37%, genotype 4 in 0.4%, genotype 5 in 0.09%, and indeterminate genotype result were found in 1.18%. Abbott assay could not identify 12 samples of genotype 3 (1.18%) and gave the indeterminate result. It also fails to assign some of the samples of genotype 1 into 1a and 1b. The indeterminate genotypes were resolved by sequencing followed by phylogenetic analysis. Genotype 3 is the predominant genotype and significantly higher in females as compared with males. Genotype 1a is more common in males than in females. Indeterminate HCV genotypes on sequencing analysis identify as genotype 3a and likewise subtype of genotype1 as 1a.

Evaluation of Three Rapid Screening Tests for Detection of Hepatitis C Antibodies on Mass Scale.

Hepatitis C virus (HCV) is a leading health problem across the globe. Only 20% of HCV positive individuals know their positive disease status. Effective HCV screening tests are required to screen both general and high-risk populations and identify the silent cases of HCV. In this study, we analyzed the performance of three rapid HCV screening kits. A total of 300 subjects from three populations groups, were enrolled from Rawalpindi and Islamabad cities of Pakistan. The three groups were blood donors (n = 50), pregnant women (n = 50), and hepatitis C positive individuals (200). Blood samples of all the individuals were screened on three rapid screening tests for anti-HCV: CTK Biotech's OnSite HCV Ab Rapid Test, SD Bioline One Step anti-HCV test, and Intec Products Advanced Quality Rapid Anti-HCV Test. The performance of these three rapid tests was also compared with the Roche Anti-HCV II test performed on the cobas 601 platform based on the electrochemiluminescence immunoassay principle. In total, 300 samples were analyzed in this study, out of which 208 were positive for anti-HCV positive and 92 were negative for anti-HCV. The sensitivities of the Intec product, SD Bioline, and CTK Biotech were 98.56%, 97.59%, and 95.67%, respectively. The specificity of SD Bioline and CTK Biotech were 100%, whereas Intec products showed 98.91% specificity. The positive predictive value (PPV) of SD Bioline and CTK Biotech was 100%, but Intec products showed 99.51% PPV. The negative predictive values of the Intec product, SD Bioline, and CTK Biotech were 96.80%, 94.84%, and 91.09%, respectively. There is a dire need to speed up HCV screening to achieve the targets in the World Health Organization global viral hepatitis strategy (2016-2021). The rapid tests evaluated in this study can be used in hepatitis screening on much larger scales.

Predictors of Therapeutic Outcome to Nucleotide Reverse Transcriptase Inhibitor in Hepatitis B Patients.

Hepatitis B is a clinically important public health issue. Infection leads to hepatocellular carcinoma. Therefore, patients need antiviral therapy for prolonged period to prevent the complication of the disease. Data concerning chronic hepatitis B (CHB) patients with high hepatitis B virus (HBV) DNA are limited. The aim of the study was to check the efficacy of the nucleoside reverse transcriptase inhibitors (tenofovir) in terms of suppression of HBV DNA. The secondary end point in the study is to evaluate trends of predictive variables that predict outcome of treatment. In this specific study, we evaluated 140 CHB male and female patients, of these 110 completed 48 weeks of treatment. On the basis of hepatitis B e antigen (HBeAg), patients were stratified; HBV DNA and hepatitis B surface antigen (HBsAg) levels were measured along with liver function tests. All enrolled patients were given tenofovir disoproxil fumarate 300 mg daily before breakfast. Overall, 69.1% of patients showed virologic response. HBeAg-negative patient group showed 68% viral suppression and HBeAg-positive patient group showed 45.9% over 24 months of treatment, while at 48 months it was shown to be 76.7% and 54.1%, respectively. None of the patients suffered HBsAg loss during the 48 months. Baseline high HBV DNA level was found as a significant predictor of response (OR, 1.9; 95% CI = 1.23-3.9, p = 0.005). None of the patients observed had serious adverse events. Mutations in the RT region of polymerase gene are shown to be associated with resistance to antiviral drugs. Among patients suffering with chronic HBV infection, HBeAg-negative patient group have better virologic response as compared with HBeAg-positive group. Higher concentration of HBV DNA at baseline has negative prediction for sustained viral suppression. The A-B motif interdomain rtL122F mutation was found in nonresponder patients in our study. Another mutation rtN248H observed in E motif considered to have effect on DNA primer grip, which forms part of binding pocket.

Analysis of Host and Viral-Related Factors Associated to Direct Acting Antiviral Response in Hepatitis C Virus Patients.

Hepatitis C virus (HCV) signifies an important health issue as it is a globally prevalent pathogen and poses a great threat to human health. Direct antiviral therapy became a landmark in treatment against chronic HCV infection as they have proven to increase sustained virological response (SVR) rate, provide shortened and simplified regimens. This study aimed to evaluate efficacy of Sofosbuvir and classify factors of treatment success and their function in therapy continuation decision. We studied host and viral factors in 310 patients who received Sofosbuvir (DAA) at a dose of 400 mg daily along with ribavirin 10 mg/kg body weight for 24 weeks and followed by 12 weeks after completion of treatment. A total of 302 patients (97.42%) showed end-of-treatment response. However, 8 patients (2.58%) were virological nonresponders and 286 patients (94.70%) achieved SVR. Among 310 HCV positive, the percentage of viral genotype 3-infected patients was 89.6%, whereas only 10.32% were affected by HCV genotype 1. Multivariable analysis showed that baseline HCV RNA (≤8 × 106 IU/mL vs. >8 × 106 IU/mL) [95% confidence interval (CI), odds ratio (OR) 3.0, 2.0 to 8.4; p = 0.004], age (<50 vs. ≥50 years) (95% CI, OR 1.9, 3.0 to 10.1; p = 0.03), liver texture (normal liver vs. coarse) are related to virological response. None of the patients included in the study has S282T substitution in NS5b of HCV. No significant difference in response was observed in patients with different genotypes of rs12979860. Liver texture, age, and viral load are predictors of SVR. In this real-life data no substitution of S282T in NS5b was observed related to failure of therapy. Oral Sofosbuvir generally has a high tolerance and can effortlessly beat the safety concerns of past regimens.

A case of a patient infected with a hepatitis C virus genotype 3a multidrug resistant variant in Pakistan.

BACKGROUND: Approximately 10 million people in Pakistan are infected with the hepatitis C virus (HCV). Most patients develop chronic hepatitis, with rare cases of spontaneous clearance. However, little is known about multidrug resistant viral variants in Pakistan. FINDINGS: This case study describes a 47-year-old male diagnosed with chronic HCV genotype 3a infection in 2003. After an initial diagnosis of viral infection, the patient remained treatment naïve for 5 years. He received two therapy cycles of interferon (IFN) plus ribavirin (RBV) in 2007 and 2010, however, he was non-responsive to the therapy. The patient then received an additional two treatment cycles of pegylated IFN α-2b plus RBV (in 2011 and 2013); he was still non-responsive. In 2016, the patient underwent sofosbuvir plus RBV combination therapy, however, the sustained virological response was still not achieved. The host genetic factor was found to be heterozygous guanine and thymine (GT) and cytosine and thymine (CT) genotypes of rs8099917 and rs12979860 polymorphism of IL28B, respectively. Phylogenetic analysis suggests that the resistant variant belong to an out-group and may require triple therapy. CONCLUSIONS: This is the first case that reports on a HCV-infected individual who was a non-responder to multiple IFN therapies in Pakistan. Further studies are needed to understand multidrug-resistant HCV variants in the Pakistani population.

Signaling cascades in thyroid cancer: Increasing the armory of archers to hit bullseye.

Thyroid cancer is a multifaceted and therapeutically challenging disease and rapidly accumulating experimentally verified findings have considerably improve our understanding of the molecular mechanisms which underlie its development. Substantial fraction of information has been added into existing landscape of molecular oncology and we have started to develop a sharper understanding of the underlying mechanisms of thyroid cancer. Wealth of information demystified different intracellular signaling cascades which are frequently deregulated in thyroid cancer. In vitro assays and xenografted mice based studies have helped us to identify drug targets and different synthetic and natural products are currently being tested to effectively treat thyroid cancer. Cabozantinib and vandetanib have been approved to treat medullary thyroid cancer (MTC) and two agents (lenvatinib and sorafenib) are also being used to treat radioactive-iodine refractory differentiated thyroid cancer. This review comprehensively summarizes most recent advancements in our knowledge related to dysregulated intracellular signaling cascades in thyroid cancer and how different proteins can be therapeutically exploited. (1) We discuss how loss of TRAIL mediated apoptosis occurred in thyroid cancer cells and how different strategies can be used to restore apoptosis in resistant cancer cells; (2) We provide detailed account of seemingly opposite roles of NOTCH signaling in thyroid cancers; (3) TGF/SMAD mediated signaling also needs detailed research because of context dependent role in thyroid cancer. Researchers have only begun to scratch the surface of how TGF signaling works in thyroid cancer and metastasis; and (4) Role of SHH signaling in thyroid cancer stem cells is also well appreciated and targeting of SHH pathway will be an important aspect in treatment of thyroid cancer. Better concepts and improved knowledge will be helpful for clinicians in getting a step closer to individualized medicine.

Human papillomavirus infection in females with normal cervical cytology: Genotyping and phylogenetic analysis among women in Punjab, Pakistan.

BACKGROUND: Globally, cervical cancer is the fourth most common cancer in women and the seventh most common cancer overall, accounting for an estimated 300 000 annual deaths. Human papillomavirus (HPV) is the second most common cause of cervical cancer worldwide. HPV screening is not a common practice in Pakistan. The aim of this study was to determine the prevalence of HPV and HPV types in women with a normal cytology of the cervix living in the upper and lower regions of Punjab, Pakistan, and to analyze the risk factors for HPV in this region. METHODS: PCR analysis was performed for 1011 female patients with a normal cytology of the cervix from various districts of Punjab Province, Pakistan. Risk factors for the acquisition of HPV were studied. High-risk HPV types (HPV16 and HPV18) were detected using the Abbott Real Time HR HPV test. To determine the genotype, partial L1 region sequences of HPV-positive samples were subjected to sequencing using MY/09/MY11 primers, and a phylogenetic tree was constructed using CLC software. RESULTS: The study found a 4.74% prevalence of HPV, with the most frequent HPV type found being the low-risk HPV6 (in 25% of infected individuals), followed by HPV55 (22.9%), HPV11 (20.8%), and high-risk types HPV45 (12.5%), HPV33 (8.33%), HPV18 (6.25%), and HPV16 (4.16%). Phylogenetic analysis of all HPV types in this study showed 80-99% nucleotide identity with types related to the same species. The sequences were clustered with China, India, Mexico, Iran, Slovenia, and Germany, showing the diversity in origin of the various genotypes prevalent in Pakistan. CONCLUSIONS: In this population with a normal cervical cytology, the prevalence of high-risk HPV types was very low. The major prevalent HPV genotype in Punjab Province of Pakistan was the low-risk HPV type 6, followed by HPV type 55. Sequencing of the partial L1 region suggested that the region was highly conserved in all reported sequences. This study highlights the need to conduct robust epidemiological studies in the region and to develop regular HPV screening so that the situation does not reach an alarming stage resulting in cervical cancer.

Evaluation of Temporal Virological Responses to Interferon-α-2b plus Ribavirin among Genotype 3a Hepatitis C Virus-Infected Patients.

OBJECTIVES: The present study aimed to examine the impact of rapid virological response (RVR) and early virological response (EVR) on sustained virological response (SVR) in chronic hepatitis C genotype 3a individuals. METHODS: The patients were given antiviral therapy with IFN-α-2b, 3 million units 3 times a week and 800-1,200 mg of ribavirin daily adjusted to the patient's body weight (<60 kg 800 mg day-1, and >60 kg 1,200 day-1). The patients received this combination therapy for 24 weeks. The patients were evaluated for their viral load at week 4, 12, and 24 using RT-PCR. RESULTS: Out of 1,471 patients, 43.3% showed a negative viral load in week 4, demonstrating RVR, whereas 56.6% maintained a high viral load. These were further separated based on viral reduction in their plasma: either negative for HCV-RNA at week 12 (n = 575), manifesting EVR, or showing a 2-log reduction in HCV viral load classified as partial EVR (PEVR; n = 259). The PEVR response was less (29.7%) compared with RVR (85.9%) and EVR (69.0%), although nonresponders were found in both groups. CONCLUSIONS: Individuals incompliant with their treatment who have a higher RVR significantly influence their SVR towards a better remission that can be treated within a short duration with standard treatment.

Prediction of promiscuous T cell epitopes in RNA dependent RNA polymerase of Chikungunya virus.

OBJECTIVE: To explore RNA dependent RNA polymerase of Chikungunya virus (CHIKV) and develop T cell based epitopes with high antigenicity and good binding affinity for the human leukocyte antigen (HLA) classes as targets for epitopes based CHIKV vaccine. METHODS: In this study we downloaded 371 non-structural protein 4 protein sequences of CHIKV belonging to different regions of the world from the US National Institute of Allergy and Infectious Diseases (NIAID) virus pathogen resource database. All the sequences were aligned by using CLUSTALW software and a consensus sequence was developed by using Uni Pro U Gene Software version 1.2.1. Propred I and Propred software were used to predict HLA I and HLA II binding promiscuous epitopes from the consensus sequence of non-structural protein 4 protein. The predicted epitopes were analyzed to determine their antigenicity through Vaxijen server version 2.0. All the HLA I binding epitopes were scanned to determine their immunogenic potential through the Immune Epitope Database (IEDB). All the predicted epitopes of our study were fed to IEDB database to determine whether they had been tested earlier. RESULTS: Twenty two HLA class II epitopes and eight HLA class I epitopes were predicted. The promiscuous epitopes WMNMEVKII at position 486-494 and VRRLNAVLL at 331-339 were found to bind with 37 and 36 of the 51 HLA class II alleles respectively. Epitope MANRSRYQS at position 58-66 and epitopes YQSRKVENM at positions 64-72 were predicted to bind with 12 and 9 HLA II alleles with antigenicity scores of 0.754 9 and 1.013 0 respectively. Epitope YSPPINVRL was predicted to bind 18 HLA I alleles and its antigenicity score was 1.425 9 and immunogenicity score was 0.173 83. This epitope is very useful in the preparation of a universal vaccine against CHIKV infection. CONCLUSIONS: Epitopes reported in this study showed promiscuity, antigenicity as well as good binding affinity for the HLA classes. These epitopes will provide the baseline for development of efficacious vaccine for CHIKV.

Prevalence of hepatitis C in people who inject drugs in the cities of Rawalpindi and Islamabad, Pakistan.

Pakistan has the second highest burden of hepatitis C (HCV) in the world. The major route of HCV transmission is contaminated blood or needle sharing. Seventy percent of people who inject drugs (PWIDs) shared needles at some time in their addiction history. The aim of the present study was to estimate the prevalence of HCV in PWIDs in cities of Pakistan. We enrolled 100 PWIDs from the Rawalpindi and Islamabad cities of Pakistan. Blood samples were taken in collection tubes and were subjected to HCV screening by using three rapid HCV screening kits including one step anti-HCV test, onsite HCV Ab rapid test and advance quality rapid anti-HCV test. All 100 blood samples were also subjected to HCV detection by using Elecsys anti-HCV II performed on the Roche Cobas 601 platform based on the ECLIA principle. Seventy-two percent of PWIDs showed the presence of HCV antibodies using the Roche anti-HCV II ECLIA test. We also compared the performance of different rapid kits in comparison with the anti-HCV II by Roche. The sensitivity of CTK kit was 84.72%, which was almost equal to the sensitivity by the SD Bioline HCV and Advanced Quality Rapid HCV tests, which was 83.33%. All three kits showed 100% specificity and positive predictive values. The results showed that the three market competitors of HCV rapid test showed almost equal results. The prevalence of HCV is very high in PWIDs in the capital twin cities of Pakistan. There is dire need to initiate the administration of a hepatitis test and treatment program for both high-risk and the general HCV-positive population. This is the optimal way to achieve HCV control targets established by the United Nations Sustainable Development Goals and Global Health Sector Strategy by WHO.

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.

Zika virus: Global health challenge, threat and current situation.

ZIKV has emerged as grave global health issue in the past few years. ZIKV was firstly isolated in 1947 from a rhesus sentinel monkey in the Zika forest in Uganda. It is usually transmitted by the bite of infected mosquitoes and infects skin fibroblasts, skin keratinocytes, etc. ZIKV until now was under reported because of its clinical similarity with the dengue and chikungunya. It is usually spread through the course of the sylvatic cycle. In this cycle, the virus or pathogen lifespan is spent between the wild animal and vectors. The intrinsic incubation period is not yet fully known but it is observed that the very first symptoms of ZIKV infection can appear or develop within 3-12 days of time period and usually subside within 7 days of time. There is a strong relationship between prenatal Zika virus infection and microcephaly; other serious brain anomalies to the infant or newborn are Guillain-Barré syndrome. To date no vaccines are available for ZIKV prevention hence only symptomatic treatment is recommended in infected patients. Usually ZIKV is detected by serologic (IgM ELISA), plaque reduction neutralization test (PRNT) along with in-house" molecular techniques (RT-PCR). ZIKV infection being imminent global health issue warrants strong protective measures to prevent it from becoming an epidemic. Early detection and prevention is the key to tackle this grave potential health hazard. J. Med. Virol. 89:943-951, 2017. © 2017 Wiley Periodicals, Inc.

Optimum predictors of therapeutic outcome in HCV patients in Pakistan.

Hepatitis C virus (HCV) constitutes a major public health issue in Pakistan. Interferon α and ribavirin is used widely in routine practice in HCV infected patients in Pakistan.Treatment prediction is an important tool in therapy management. The present study aims to evaluate trends of predictive variables of treatment outcome in patients with different genotypes. The analysis comprised of 921 patients infected with different HCV genotypes. All the patients received IFN α-2b combined with ribavirin for 24 weeks. Overall, 60.2% patients achieved Sustained virologic response (SVR). In females sustained virologic response (SVR) was higher in age group <40 years (77.2%) than ≥40-50 years (60%) but in male SVR was almost equal in both age groups. We also found higher SVR with low pretreatment viral load (72.4%, P < 0.0001). Sustained Virologic Response in genotype 3a was 63.1%, 3b was 55%, 1a was 36.3% and 1b was 35% 3a +3b was 55.0% and 1a+3a was 42.9%. According to multivariable logistic regression analysis age < 40 years (2.0; 95%CI, 1.49-2.84; P = 0.0001), low pretreatment RNA level<800,000 IU/ml (4.0; 95%CI, 2.64-6.17; P = 0.0001), early virologic response at week 12 (12.3; 95%CI, 8.18-18.58; P < 0.0001) and non-fatty liver (2.5; 95%CI, 3.6-6.2; P = 0.005) showed significance for SVR. Nucleotide substitution in 5'UTR before treatment failed to show any characteristic pattern that has correlation with sustained response. Subtype 3a showed 95% presence among patients with age <40 years while older patients showed 79.9%.

Polymorphism of the IL28B gene (rs8099917, rs12979860) and virological response of Pakistani hepatitis C virus genotype 3 patients to pegylated interferon therapy.

BACKGROUND: The gold standard treatment for chronic hepatitis C virus (HCV) infection is pegylated interferon (PEG-IFN) in combination with ribavirin. Most patients treated with PEG-IFN achieve a sustained virological response (SVR). However host genetic factors play a vital role in the spontaneous and treatment-induced clearance of HCV infection from these infected patients. In the current study, polymorphisms of IL28B (rs8099917 and rs12979860) were analyzed and their association with the virological response to PEG-IFN alpha treatment was determined. METHODS: One hundred and fifty HCV genotype 3 patients were assessed to study the correlation of IL28B with a therapeutic regimen of PEG-IFN alpha plus ribavirin. Twenty patients were excluded due to a refusal to participate in the study and 25 patients failed to meet the inclusion criteria. Of the 105 patients recruited, 49 (46.7%) were male and 56 (53.3%) were female. In order to determine single nucleotide polymorphisms of rs8099917 and rs12979860, the sample was amplified by PCR and then IL28B typing was carried out by restriction fragment length polymorphism (RFLP) followed by standard sequencing. RESULTS: We found three types of genotype in rs8099917 of IL28B: wild-type TT in 60.0% of patients, heterozygous GT minor genotype in 36.2%, and GG in 3.8%. The frequency of the CC genotype of rs12979860 was 54.3%, CT was 37.1%, and TT was 8.6%. Overall, SVR was achieved in 68.6% of patients. A higher SVR was achieved for patients with the favorable genotype CC of rs12979860, with 84.2% as compared to 56.4% and 22.2% for minor genotype CT and TT, respectively (p=0.0001). We did not find a significant association for SVR to antiviral treatment in patients with genotype TT (rs8099917) (71.9%, p=0.36). The rapid virological response (RVR) rate was significantly higher in patients with major genotype TT (88.9%, p=0.04). These results show that IL28B polymorphism is highly associated with SVR to therapy in the Pakistani population infected with HCV genotype 3. CONCLUSIONS: HCV-infected patients carrying homozygous C/C have a higher chance of SVR. In addition, patients who carry T/T (rs8099917) have a higher chance of RVR.

Metabolic syndrome is associated with poor treatment response to antiviral therapy in chronic hepatitis C genotype 3 patients.

INTRODUCTION: Hepatitis C viral (HCV) infection is caused by an RNA virus. HCV infection is considered to induce systemic disease that causes steatosis, alters lipid metabolism, and results in metabolic syndrome. This study aimed to investigate the therapeutic outcome in HCV genotype 3 patients with metabolic syndrome. MATERIALS AND METHODS: A total of 621 HCV-positive patients who visited the hospital for treatment were screened. Among these, 441 patients were enrolled for antiviral therapy. These enrolled patients were assessed for metabolic syndrome according to the International Diabetes Federation criteria. Group A included patients with metabolic syndrome and group B included patients without metabolic syndrome. All patients received peginterferon-α2a (180 µg/week) and ribavirin (10 mg/kg/day) for 6 months. RESULTS: The prevalence of metabolic syndrome in chronic HCV patients was 37.9%. We observed that metabolic syndrome was more common among female compared with male participants (43.9 vs. 28.8%, P=0.005). It was found that sustained virologic response (SVR) rates were significantly higher in the patients in group B (without metabolic syndrome) compared with the patients in group A who had metabolic syndrome (72.2 vs. 43.7%, P<0.05). Older patients were at a higher risk for metabolic syndrome and a correlation of metabolic syndrome with nonresponse to antiviral therapy was observed. An interesting correlation among metabolic syndrome, age, and SVR was found: with age, SVR decreases, while metabolic syndrome increases. CONCLUSION: Metabolic syndrome has an influence on therapeutic outcomes in terms of SVR. Moreover, this information can identify patients who might have a low chance of attaining an SVR and a timely decision may protect the patients from the adverse effects of therapy.

Transcript analysis of P2X receptors in PBMCs of chronic HCV patients: an insight into antiviral treatment response and HCV-induced pathogenesis.

BACKGROUND: After invasion of hepatocytes and immune cells, hepatitis C virus has the ability to escape from the host immune system, leading to the progression of disease into chronic infection with associated liver morbidities. Adenosine 5'triphosphate (ATP) is released in most of the pathological events from the affected cells and acts as a signaling molecule by binding to P2X receptors expressed on the host's immune cells and activates the immune system for pro-inflammatory response. Therefore, the present study was designed to analyze the transcript expression of the ionotropic purinergic P2X receptors on peripheral blood mononuclear cells (PBMCs) of chronic HCV patients to have study the immune responses mediated by P2X receptors in chronic HCV infections. METHODS: PBMCs were isolated from the collected blood samples. Transcript analysis of P2X receptors in PBMCs was done. The identity of amplified product was confirmed by sequencing PCR, while the quantification of the transcript expression was done by real time PCR. The relative expression of the P2X receptors was analyzed by unpaired Student's t test using GraphPad Prims 5 software. RESULTS: We found that out of seven isoforms of P2X receptors, P2X1, P2X4, P2X5, and P2X7 receptors are expressed on the PBMCs. P2X1 and P2X7 are significantly upregulated in treatment-naïve chronic HCV patients by 2.2- and 2.5-fold, respectively. However, only P2X7 expression is found increased by 2.7-fold in patients achieving sustained virological response (SVR) after antiviral treatment compared to healthy controls. The expression of P2X receptors remained unaltered in chronic HCV patients not responding to the treatment. CONCLUSION: The present study confirms the significant involvement of P2X receptors in the immune responses mediated by the PBMCs in the chronic HCV infection, which should be further investigated to devise strategies to augment the immune system against this chronic viral disease.