HCV infection among Saudi population: high prevalence of genotype 4 and increased viral clearance rate.

HCV is a major etiological agent of liver disease with a high rate of chronic evolution. The virus possesses 6 genotypes with many subtypes. The rate of spontaneous clearance among HCV infected individuals denotes a genetic determinant factor. The current study was designed in order to estimate the rate of HCV infection and ratio of virus clearance among a group of infected patients in Saudi Arabia from 2008 to 2011. It was additionally designed to determine the genotypes of the HCV in persistently infected patients. HCV seroprevalence was conducted on a total of 15,323 individuals. Seropositive individuals were tested by Cobas AmpliPrep/Cobas TaqMan HCV assay to determine the ratio of persistently infected patients to those who showed spontaneous viral clearance. HCV genotyping on random samples from persistently infected patients were conducted based on the differences in the 5'untranslated region (5'UTR). Anti-HCV antibodies were detected in 7.3% of the totally examined sera. A high percentage of the HCV infected individuals experienced virus clearance (48.4%). HCV genotyping revealed the presence of genotypes 1 and 4, the latter represented 97.6% of the tested strains. Evidences of the widespread of the HCV genotype 4 and a high rate of HCV virus clearance were found in Saudi Arabia.

Quantitative DNA analysis of very low-level hepatitis B viremic patients reporting to the gastroenterology clinic.

OBJECTIVE: To examine data on very low-level viremic hepatitis B virus (HBV) infections in patients reporting to a gastroenterology clinic, and to investigate methods to improve analysis to avoid missing follow-up data and improve the management of HBV infection, and minimize morbidity and mortality outcomes. METHODS: A total of 104 patients with very low-level viremic HBV whom reported to the gastroenterology clinic at Al-Hada Armed Forces Hospital, Taif, Saudi Arabia and had a reading of <12 IU/mL on the real time (RT) polymerase chain reaction (PCR) detection system were enrolled in this study. For serological testing (for example, hepatitis B surface antigen [HBsAg]), we examined patients' results recorded in the laboratory information system since early 2007. Liver enzymes, alanine aminotransferase, and aspartate aminotransferase were assessed in some cases. RESULTS: After analyzing the data collected from 1,178 patients, we found 104 (8.83%) cases that fit the criteria for our study, including a reading of <12 IU/mL. We formed 6 groups of participants based on HBsAg reactivity and very low, elevated, or no viremia, and found 4 cases of continuous occult hepatitis B infection. CONCLUSION: The very low levels of DNA found had a diagnostic impact on the management of HBI and yielded several suggestions for clinicians regarding follow-up with patients. It is important to use a sensitive RT PCR to monitor the course of HBV infection.

The role of caspase activation and mitochondrial depolarisation in cultured human apoptotic eosinophils.

Caspases are key intracellular molecules in the control of apoptosis, but little is known concerning their relative contribution to the cascade of events leading to eosinophil apoptosis. We examined caspase-3, -8, and -9 activities in receptor ligation dependent apoptosis induction in the cultured eosinophils (CE). CE cultured alone for 48 hours exhibited constitutive apoptosis (12% ± 1.2). Significant (P < 0.05) enhancement of eosinophil apoptosis was observed following monoclonal antibody (Mab) treatment with CD45 (40% ± 0.7), CD95 (36% ± 1.6), or CD69 (34% ± 0.2). Caspase activity was analysed using the novel CaspaTagTM technique and flow cytometry. CE ligated with CD45 (Bra55), CD95 (Fas) and CD69 Mab resulted in caspase-3 and -9 activation after 16 hours post-ligation. This trend in caspase-3 and -9 activation continued to increase significantly through to the 20 and 24 hours time points when compared to isotype control. Activated up-stream caspase-8 was detected 16 and 20 hours after treatment with CD45, CD95 and CD69 Mab followed by a trend toward basal levels at 24 hours. Ligation of CD95 was followed by mitochondrial permeabilization, as demonstrated by marked increase in mitochondrial transmembrane potential ([Formula: see text]) at all time points. However, ligation with CD45 and CD69 failed to induce a change in [Formula: see text] at 16 hours post-treatment compared to isotype control even though there was an alteration in mitochondrial downstream-caspase activity following ligation with these Mab(s) at this time point. At 20 and 24 hours post-ligation, CD45 or CD69 induce significantly altered levels of [Formula: see text]. Thus, the intrinsic and extrinsic caspase pathways are involved in controlling receptor ligation-mediated apoptosis induction in human eosinophils, findings that may aid the development of a more targeted, anti inflammatory therapy for asthma.

Nucleic acid amplification technology for hepatitis B virus, and its role in blood donation screening in blood banks.

OBJECTIVE: To investigate the performance of hepatitis B virus polymerase chain reaction (HBV PCR) using one of the commercial methods used around the world to screen for HBV in some blood donors where other conventional serological assays have limitations to detect the virus. METHODS: This study was designed to use Amplicor AmpliScreen for HBV testing to detect the presence of the HBV DNA in the specimens tested by COBAS AmpliPrep system using a modified manufacture protocol COBAS AmpliPrep of total nucleic acid isolation (TNAI) kit. All serological tests were carried out on the donors' samples to detect the hepatitis B surface antigen (HBsAg), Australian antibody anti-HBs (AUSAB) and hepatitis B core antigen (HBcAg) in the 2 periods of the study. The first period was started in February 2005 and the second period was started in April 2007. Both periods were continued for 2 months after beginning in the molecular pathology laboratory, Al-Hada Armed Forces Hospital, Taif, Kingdom of Saudi Arabia. The 600 donors' data were then studied and analyzed. RESULTS: Five nucleic acid amplification test (NAT-HBV) positives were found out of 600. There were 3 positive for HBcAb and negative for HBsAg, 2 had reading with <100 mIU/mL anti-HBs (AUSAB), and one had >100 mIU/mL AUSAB readings. CONCLUSION: Our results show that there is a possibility to have occult HBV infection in some donors that cannot be detected by the HBsAg routine serological assays. Moreover, the study can be useful to formulate a new deferral policy based on the implementation of NAT-HBV for blood screening.

A close link between Fas, p53 and Apaf-1 in chronic myeloid leukemia.

In chronic myeloid leukemia (CML) proliferation is increased and resistance to apoptosis has been proposed as a mechanism accounting for myeloid cell expansion. There is still controversy on whether apoptosis plays an important role in the regulation of myelopoiesis. This study aims to investigate whether apoptosis-related proteins play a role in the evolution of CML and to identify, the relationship between Fas, p53 and apoptosis protease activating factor (Apaf-1) in CML. We found increased p53 and Apaf-1 messenger ribonucleic acid (mRNA) in patients with CML. However, one patient, who had a p53 point mutation, showed a massive elevation of p53 mRNA during blast crisis yet, conversely, a considerable reduction in Apaf-1 mRNA and Fas mRNA. Our results show an in-vivo linkage between Fas, p53 and Apaf-1 transcription regulation. This suggests that key genes involved in apoptosis are also involved in CML disease progression.

Nucleic acid amplification technology screening for hepatitis C virus and human immunodeficiency virus for blood donations.

OBJECTIVE: To investigate the performance of the commercial Roche COBAS AmpliScreen assay, and demonstrate whether the COBAS AmpliScreen human immunodeficiency virus-1 (HIV-1) test, v1.5, and COBAS AmpliScreen hepatitis C virus (HCV) v 2.0 for screening for HIV-1 and HCV RNA in the donated blood units from which plasma mini pools were collected, by nucleic acid amplification technology (NAT), could detect the positive pools and reduce the risk of transmission of infections for those routinely tested by serological assays. METHODS: The study was performed on 3288 plasma samples collected from blood donors in a period of 13 months, from August 2004 to August 2005, at Al-Hada Armed Forces Hospital, Molecular Pathology Laboratory, Taif, Kingdom of Saudi Arabia. The samples were tested by the reverse transcriptase polymerase chain reaction (RT-PCR) after RNA extraction (this represents the major method in NAT assays), in parallel with the routine serological testing to detect qualitatively for HIV-1 and HCV. RESULTS: The NAT assays that include an automated COBAS AmpliPrep system for RNA extraction and COBAS Amplicor Analyzer using AmpliScreen kits for RT-PCR assays, and the routine serological screening assays for the detection of the HIV-1 and HCV RNA in the plasma samples from the blood donors have shown to be a reliable combination that would meet our requirements. The collected data further confirms the results from the serological assays and enables us to decrease the residual risk of transmission to a minimum with the finding of no seronegative window period donation. The results demonstrate that out of 3288 samples, the percentages of RT-PCR (NAT) negative blood donations that were also confirmed as seronegative were 99% for HCV, and 99.1% for HIV-1. CONCLUSION: The modified combined systems (automated COBAS AmpliPrep system for RNA extraction and COBAS Amplicor Analyzer using AmpliScreen kits for RT-PCR assays) for NAT screening assays has allowed the release of all blood donations supplied in the specified period of the study with no seronegative window period donations. This facilitates keeping the residual risk of transmission of HIV-1 and HCV to its minimum through blood transfusion.

Apoptosis role of FAS/FAS ligand system in the regulation of myelopoiesis.

OBJECTIVES: CD34+ cells and colony forming unit-granulocyte and macrophage (CFU-GM) from human bone marrow were used to investigate the role of Fas/FasL system in the regulation of myelopoiesis. METHODS: Fas and FasL expression in CD34+ cells and in day 14 CFU-GM were measured by RT-PCR and immunofluorescence respectively. The functional assays for the CFU-GM were measured by a standard colony assay and the proliferative capacity of CFU-GM was measured by replating the primary colony and observing the secondary colony formation. Human marrow cells were treated with IETD (caspase-8 inhibitor) or anti-Fas CH-11 Mab. RESULTS: Treatment with the CFU-GM with IETD significantly increased, the proliferative capacity, while anti-Fas CH-11 Mab markedly reduced it. Fas and FasL expression were demonstrated using RT-PCR and immunofluorescence respectively. CONCLUSION: Fas, FasL, and caspase activation are likely to play an important role in the regulation of myelopoiesis.

An assessment of a multiplex PCR assay for differentiating clinically important mycobacteria based on pncA gene variation.

The pncA genes in mycobacteria are responsible for the production of pyrazinamidase (PZase). In Mycobacterium tuberculosis, PZase hydrolyses pyrazinamide (PZA) to pyrazonic acid, a compound that possesses bactericidal activity against tubercle bacilli. Nucleotide sequences of pncA genes found within mycobacteria where aligned in an effort to ascertain the significance of any variability in sequence. Three sets of primers (one degenerate and five consensus sequences) were designed and employed in a multiplex PCR assay to amplify the pncA region in seven clinically common mycobacteria. The banding patterns generated from each species in conjunction with PZase activity tests demonstrated that the mycobacterial species examined could be clearly identified and differentiated from one another. Although not yet tested with clinical isolates, the combination of these two assays has provided a promising discriminatory tool for the identification of commonly encountered clinical mycobacteria species.