Over the past decade, many authors have focused on PCR as a powerful technique for the evaluation of human cytomegalovirus (CMV). The key to PCR lies in the design of oligonucleotides, as the specific sequences largely affect PCR's efficacy and sensitivity. This study was designed to examine the quality of published sequences of CMV primers and probes.PubMed was searched in the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov) for English peer-reviewed articles using CMV and PCR as keywords. Articles reporting on virus genotyping or species-level identification, as well as letters to editors and reviews were excluded. The full texts of 91 papers published between 1993 and 2004 were studied. Of these, 34 papers did not describe the detailed nucleotide sequence, including 17 papers using commercially available kits. The remaining 57 papers with a total of 199 CMV-specific oligonucleotides were examined. Oligonucleotides with identical sequences or with one additional nucleotide at either the 3Ј or 5Ј end of the sequence were identified as synonymous.Using The Sequence Manipulation Suite web-based programs (written by Paul Stothard, University of Alberta, Canada), the binding sites of all 199 oligonucleotides were identified using GenBank strain AD169 genome sequence (GenBank accession no. X17403 and NC_001347). Mismatches to all published sequences of CMV were analyzed by the Basic Local Alignment Search Tool (BLAST) (http://www .ncbi.nlm.nih.gov/BLAST/). Papers containing oligonucleotide errors were studied for technical reasons for employment of mutated oligonucleotides or subsequently published errata. Moreover, information on identified errors was conveyed to all corresponding authors and coauthors.Ten oligonucleotides did not match any CMV strain, and reasons were specified neither in the respective articles nor in authors' replies (Table 1). Primers R2, F6, and F9 were incorrectly transferred from previous publications (2, 5, 7) as indicated by the authors (V. H. Aquino and X. L. Pang, personal communication). The oligonucleotides F1, R3, and P10 were apparently also incorrectly transferred from prior publications (14). Moreover, primers F4 and R5 possessed mismatches at their 3Ј-end triplets, which may reduce PCR efficiency (21). Furthermore, the two degenerated probes (P7 and P8) contained Y (C/T) instead of R (A/G) in P7 and vice versa in P8. Obviously, these mismatches may drastically affect the efficacies of these probes (17).In summary, we show that 5% of the CMV oligonucleotides included mismatches to all published sequences half of which were incorrectly reported mostly in secondary publications. Moreover, since we considered all sequences correct when they matched at least one CMV strain, even when the authors used a different strain for design or admitted an error, the number of errors is underestimated by our approach. These observations point to the possibility that the report of erroneous primers and probes is a widespread problem irritating other researchers. I...

Changes in the copy numbers of cell-free nuclear DNA (cf-nDNA) and cell-free mitochondrial DNA (cf-mtDNA) have shown promising diagnostic utilities among patients with head and neck squamous cell carcinoma (HNSCC). Considering the absence of objective prognostic tools for HNSCC surveillance, this study aimed to assess the utility of saliva-based cf-nDNA and cf-mtDNA in predicting the overall survival of patients with HNSCC. The study included ninety-four patients with a confirmed HNSCC diagnosis with a mean follow-up time of 32.04 months (±19.1). A saliva-based liquid biopsy was collected from each patient. A multiplex quantitative PCR was used to determine the absolute number of cf-nDNA and cf-mtDNA. The Kaplan–Meier estimator and Cox proportional hazards regression models were used to assess overall survival. The absolute copy numbers of cf-nDNA and cf-mtDNA were statistically significantly higher among the deceased patients than among the censored ones (p < 0.05). Individuals with elevated levels of cf-nDNA or cf-mtDNA were associated with a significantly poorer overall survival (p ≤ 0.05). A univariate analysis showed that only the absolute copy number of cf-mtDNA was the sole predictor of overall survival. However, the multivariate analysis showed that all the absolute copy numbers of cf-nDNA, the absolute copy numbers of cf-mtDNA, and the stage of HNSCC were predictors of overall survival. Our study confirms that saliva is a reliable and non-invasive source of data that can be used to predict the overall survival of patients with HNSCC, where cf-mtDNA levels act as the sole predictor.

Standardization of human cytomegalovirus (CMV) PCR is highly recommended. As primer design is essential for PCR sensitivity, this study evaluated all published CMV primer pairs to identify the most sensitive for single-round real-time PCR. PubMed (1993PubMed ( -2004 was searched for original papers aimed at CMV PCR. Fifty-seven papers were identified revealing 82 different primer pairs. Of these, 17 primer sets were selected for empirical study, as they were either used in real-time PCR or were evaluated comparatively by conventional PCR. After optimizing the PCR conditions, these primer sets were evaluated by real-time PCR using a SYBR Green format. Analytical sensitivities were assessed by testing the reference standard CMV strain AD169. A BLAST search was performed to identify mismatches with published sequences. Additionally, 60 clinical samples were tested with the three primer sets showing highest analytical sensitivity and the best match to all CMV strains. Three primer sets located in the glycoprotein B (UL55) gene region were found to be the most sensitive using strain AD169. However, two of these showed a considerable number of mismatches with clinical isolates in a BLAST search. Instead, two other pairs from the lower matrix phosphoprotein (UL83) gene and DNA polymerase (UL54) gene showed reasonable sensitivity and no mismatches with clinical isolates. These three pairs were further tested with clinical samples, which indicated that the two primer sets from UL55 and UL54 were the most sensitive. Interestingly, the analytical sensitivity of the PCR was inversely correlated with the size of the PCR product. In conclusion, these two primer pairs are recommended for a standardized, highly sensitive, real-time PCR. INTRODUCTIONReal-time PCR is widely considered as an efficient and highly sensitive technique for the evaluation of human cytomegalovirus (CMV) DNA kinetics (Hong et al., 2004;Kearns et al., 2001;Piiparinen et al., 2004;van Doornum et al., 2003), provided that the target sequence and primers are properly selected (Caliendo et al., 2000;Gault et al., 2001). To date, several evaluation studies have demonstrated significant differences in sensitivity of various in-house and commercial PCRs (Allen et al., 1995;Boeckh et al., 2004;Distefano et al., 2004;Weinberg et al., 1998;Zweygberg-Wirgart et al., 1998), which hinder the conduction of comparative studies (Caliendo et al., 2000;Tong et al., 2000) and accurate follow-up of patients (Gault et al., 2001). Moreover, failure of the most frequently used primers to detect some CMV isolates (Allen et al., 1995;Distefano et al., 2004;Zweygberg-Wirgart et al., 1998), and hence contradictory results between different laboratories (Bustin, 2005;Razonable et al., 2002), has been reported. All of these observations imply that the most appropriate target region for amplification has not yet been defined (Gault et al., 2001) and that standardization is still required for reliable and comparable PCR results (Bustin, 2005;Gault et al., 2001;Tong et al., 2000). As th...

Our results suggest including anti-HBc as an additional screening test for blood donors in Syria to reduce the risk of HBV transmission. As the most cost-effective measure, anti-HBc-positive donors should be tested quantitatively for anti-HBs and only donors with no or low (<100 IU L(-1) ) anti-HBs should be deferred.

Background. In Syria, CML patients are started on tyrosine kinase inhibitors (TKIs) and monitored until complete molecular response is achieved. BCR-ABL mRNA transcript type is not routinely identified, contrary to the recommendations. In this study we aimed to identify the frequency of different BCR-ABL transcripts in Syrian CML patients and highlight their significance on monitoring and treatment protocols. Methods. CML patients positive for BCR-ABL transcripts by quantitative RT-PCR were enrolled. BCR-ABL transcript types were investigated using a home-made PCR method that was adapted from published protocols and optimized. The transcript types were then confirmed using a commercially available research kit. Results. Twenty-four transcripts were found in 21 patients. The most common was b2a2, followed by b3a2, b3a3, and e1a3 present solely in 12 (57.1%), 3 (14.3%), 2 (9.5%), and 1 (4.8%), respectively. Three samples (14.3%) contained dual transcripts. While b3a2 transcript was apparently associated with warning molecular response to imatinib treatment, b2a2, b3a3, and e1a3 transcripts collectively proved otherwise (P = 0.047). Conclusion. It might be advisable to identify the BCR-ABL transcript type in CML patients at diagnosis, using an empirically verified method, in order to link the detected transcript with the clinical findings, possible resistance to treatment, and appropriate monitoring methods.

Introduction: We aimed to evaluate the prevalence of "anti-HBc alone" among Syrian blood donors, highlighting the possibility of representing occult HBV infection. Methodology: Sera of 3,896 healthy blood donors were tested for both HBsAg and anti-HBc. HBsAg-negative, anti-HBc-positive samples were further tested for the antibodies to HBsAg (anti-HBs), and "anti-HBc alone" sera were tested for HBV DNA. Results: Of 3,830 HBsAg-negative donors, 63 were "anti-HBc alone" donors, five of whom were HBV DNA positive. Conclusions: Greater consideration should be given to the "anti-HBc alone" serological profile in blood screening, premarital testing, organ transplantation tests, and other HBV transmission-related procedures in Syria.

Background and objectivesBlood transfusion is a lifesaving therapy for patients with hemoglobinopathies. However, the need of frequent transfusion carries the risk of transmitting hepatitis B and C infections which are intermediately prevalent in Syria. Despite screening blood donations with sensitive methods, the risk of transmission is still present when infectious blood is donated within the window period. This study aimed to investigate the incidence of HBV and HCV seropositivity, and its association with multiple transfusions among Syrian hemoglobinopathies patients.Materials and MethodsHBsAg, anti-HBc, anti-HBs and anti-HCV were tested for 159 Syrian multi-transfused patients by Enzyme-Linked Immunosorbent Assay (ELISA).ResultsThirty-nine of 159 (24.5%) multi-transfused patients were HBsAg/anti-HBc or anti-HCV positive, 26 (16%) of which never visited the dentist, and they either tested postsurgically negative for HBsAg and anti-HCV or never underwent a surgical procedure. On the contrary of anti-HCV seropositivity, HBsAg/anti-HBc seropositivity was significantly associated with the number of blood transfusions, number of blood units and age (P < 0.001).ConclusionAbout one-sixth of our patients most likely acquired HBV/HCV infection via blood transfusion. Administering HBV vaccine, ensuring the immune status, and monitoring hepatitis markers might considerably minimize the incidence of viral hepatitis among multi-transfused patients.

Background: STAT4 rs7574865 polymorphism has been evidently associated with susceptibility to Rheumatoid Arthritis (RA) in European and Eastern Asian populations, whereas studies in other countries reported otherwise.Objective:We investigated the distribution of STAT4 rs7574865 polymorphism in a group of Syrian RA patients.Methods:Eighty-one RA patients and forty healthy controls were enrolled and STAT4 rs7574865 was genotyped by direct sequencing. RA patients were stratified according to Anti-Citrullinated Protein Antibodies (ACPA) status for analysis.Results:Minor T allele frequencies were 30.4%, 16.7%, and 23.8% in ACPA-positive RA patients, ACPA-negative RA patients, and healthy controls, respectively. No significant differences in STAT4 rs7574865 allele/genotype frequencies were found between ACPA-positive RA patients, ACPA-negative RA patients, and healthy controls (P>0.05).Conclusion: STAT4 rs7574865 TT genotype showed a potential impact on ACPA positivity in Syrian RA patients. However, STAT4 rs7574865 effect on RA onset and severity is minor compared to other genetic factors such as HLA-DRB1 shared epitope alleles.