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Interferon (IFN) exhibits a potent antiviral activity in vitro and plays a major role in the early defense against viruses. Like IFN, the proinflammatory chemokine, interleukin (IL)-8, is induced by viruses and appears in circulation during viral infections. In an in vitro cytopathic effect assay for IFN, we found that IL-8 can inhibit IFN-α activity in a dose-dependent manner. This action was reversed by specific monoclonal antibodies to IL-8. The chemokine was able to attenuate the IFN-mediated inhibition of viral replication as determined by measuring infectious virus yield. IL-8 also diminished the ability of IFN to inhibit an early stage of viral replication since IL-8 attenuated the inhibition of the formation of viral proteins. It appeared that IL-8 interfered with a late rather than an early step of IFN-mediated pathway such as early gene expression. The IL-8 inhibitory action on IFN-α antiviral activity was associated with reduced 2′,5′-A oligoadenylate synthetase activity, a pathway well correlative with the anti– encephalomyocarditis virus action of IFN-α. Understanding pathways that antagonize IFN action may lead to novel approaches to potentiate endogenous and therapeutic IFN.

Although polyomavirus JC (JCV) is the proven pathogen of progressive multifocal leukoencephalopathy, the fatal demyelinating disease, this virus is ubiquitous as a usually harmless symbiote among human beings. JCV propagates in the adult kidney and excretes its progeny in urine, from which JCV DNA can readily be recovered. The main mode of transmission of JCV is from parents to children through long cohabitation. In this study, we collected a substantial number of urine samples from native inhabitants of 34 countries in Europe, Africa, and Asia. A 610-bp segment of JCV DNA was amplified from each urine sample, and its DNA sequence was determined. A worldwide phylogenetic tree subsequently constructed revealed the presence of nine subtypes including minor ones. Five subtypes (EU, Af2, B1, SC, and CY) occupied rather large territories that overlapped with each other at their boundaries. The entire Europe, northern Africa, and western Asia were the domain of EU, whereas the domain of Af2 included nearly all of Africa and southwestern Asia all the way to the northeastern edge of India. Partially overlapping domains in Asia were occupied by subtypes B1, SC, and CY. Of particular interest was the recovery of JCV subtypes in a pocket or pockets that were separated by great geographic distances from the main domains of those subtypes. Certain of these pockets can readily be explained by recent migrations of human populations carrying these subtypes. Overall, it appears that JCV genotyping promises to reveal previously unknown human migration routes: ancient as well as recent.

Ninety-four strains of methicillin-resistant Staphylococcus aureus (MRSA) were collected from patients nursed in several hospitals in Saudi Arabia, before they were referred to King Faisal Specialist Hospital and Research Centre for tertiary care. The hospitals were from geographically diverse regions and as such the entirety of Saudi Arabia was covered. All strains were genetically typed by random amplification of polymorphic DNA (RAPD) analysis using three different primers and a representative subset of the strains was analyzed with pulsed-field gel electrophoresis (PFGE) as well. It was concluded that 87 out of 94 (93%) belong to a single clonally related lineage of MRSA. In the other 7 cases, the DNA banding patterns were shown to differ only slightly from those determined for the clonal type. PFGE analysis confirmed the homogeneity of the collection of strains. When the RAPD and PFGE fingerprints obtained for the Saudi clone were compared to those generated for a collection of MRSA with a more diverse geographical background, it was shown that the clonal type from Saudi Arabia was not identical to any of these MRSA strains. Our data provide another example of the capacity of certain MRSA clones to expand through entire nations and establish themselves permanently among large number of hospitals and, consequently, even larger numbers of patients.

Background Extended-spectrum β-lactamase (ESBL)-producing Klebsiella species cause worldwide problems in neonatal intensive care units (NICUs). This study aimed to determine possible risk factors for infection or colonization with ESBL-producing Klebsiella pneumoniae (ESBLKp) during an outbreak in the NICU. Methods A retrospective cohort study was conducted among neonates admitted to the NICU of a teaching hospital in Riyadh, Saudi Arabia, during an outbreak of ESBLKp from April to July 2008. The incidence density ratio was calculated to determine possible predictors of ESBLKp colonization or infection. Results During 2,265 person-days of follow-up of 118 neonates, 4 became infected, and 8 were colonized with ESBLKp. Univariate analyzes revealed that, among 14 neonates who were treated with vancomycin, 9 (64.3%) developed infection or colonization with ESBLKp, whereas, among 104 neonates who were not treated with vancomycin, 3 (2.9%) were affected, with an incidence density ratio of 4.22 (95% confidence interval: 1.47-5.15). Parenteral feeding and mechanical ventilation were found to be marginally significant risk factors. Conclusion Treatment with vancomycin appears to be a risk factor for infection or colonization with ESBLKp in the NICU setting.

MHC class I polypeptide-related chain A (MICA), mapping to 6p21.33, belongs to the non-classical class I family and its expression is induced by several stress factors including viral infection. A recent genome-wide association study has identified a single nucleotide polymorphism (SNP) of MICA, rs2596542 to be significantly associated with hepatitis C-induced hepatocellular carcinoma (HCC) in a Japanese population. Therefore, this study aims to investigate whether the SNP rs2596542 plays any role in hepatitis B virus (HBV) sero-clearance or in the development of complications associated with chronic HBV such as cirrhosis and/or HCC. TaqMan genotyping assay was used to identify the association of the SNP among 584 normal healthy controls and 777 HBV-infected patients. The patient group was further categorized into inactive carriers (Group I), active carriers (Group II), cirrhosis (Group III) and cirrhosis-HCC (Group IV). Variation at this SNP was found to be significantly more frequent in control subjects than in patients (OR = 0.852; 95% C.I. = 0.730-0.994; p = 0.0415). Also, the SNP was found to have a highly significant association when the inactive carriers were compared to the rest of the patients (OR = 1.308; 95% C.I. = 1.058-1.617; p = 0.0130). The TT genotype was found to occur more frequently among active HBV carriers (groups II, III and IV) when compared to inactive HBV carriers, thus suggesting that the rs2596542-T may be recessively associated with an active HBV infection. However, no significant association was observed in the case of HBV-related cirrhosis or HCC. These findings indicate that the MICA rs2596542 has a significant role in HBV infection.

Tuberculosis is a significant threat to human health, infecting nearly one third of the world’s population and causing over a million deaths per year. The need for an effective vaccine against tuberculosis is apparent however, the current vaccine has not been successful neither in the prevention nor recovery from infection. The pathogenesis of the causative agent, Mycobacterium tuberculosis has been extensively studied. The mechanisms of immune evasion and modulation that enable persistence of infection through subclinical latency and eventually active tuberculosis have been partially described. Many of these mechanisms are directly antagonistic to the intended benefit of vaccines, leaving a conundrum that has yet to be resolved. This review will first examine the nature of tuberculosis pathogenesis in the context of the immune response and outline the varied processes utilized by the bacterium to cause modulation. The review will also investigate other vaccination options that may overwhelm these microbial mechanisms or avert them entirely, allowing for the engagement of the immune response and clearance of the bacterium.

Infection due to community-acquired strains of methicillin-resistant Staphylococcus aureus (CA-MRSA) has been reported with increasing frequency. Herein is described the nosocomial transmission of CA-MRSA involving 13 neonates and two mothers in a well-infant nursery in a teaching hospital in Saudi Arabia.From October to November 2009, temporally related cases of CA-MRSA skin and soft-tissue infection occurred in newborns shortly after discharge from a well-infant nursery. An outbreak investigation including case identification, review of medical records, staff screening, environmental cultures, pulsed-field gel electrophoresis, and a case-control study were conducted. Controls were selected from among asymptomatic neonates admitted to the same nursery and matched for the day of admission.Fifteen subjects were found to be CA-MRSA positive: 13 neonates and two mothers. The crude attack rate among neonates was 5.5% during the outbreak period. All 13 neonates presented with skin and soft-tissue infection; one of the mothers had mastitis and a breast abscess. The source of the outbreak was not evident. Pulsed-field gel electrophoresis showed that all of the tested isolates from one strain except one, all contained the staphylococcal cassette chromosome mec (SCCmec) type IV.MRSA strains that initially emerged in the community are now causing disease in health-care settings. Adherence to standard infection control practices, including consistent hand hygiene, in newborn nurseries is important to prevent transmission in such settings.

We aimed to characterize the vancomycin genotype/phenotype, carriage of putative virulence genes, and genetic relatedness of Enterococcus faecium isolates in Saudi Arabia. E. faecium isolated from inpatients at our medical center were studied. Sensitivity to ampicillin, linezolid, teicoplanin, quinupristin/dalfopristin, tetracycline, and ciprofloxacin was determined. The presence of van genes and virulence genes for aggregation substance (Asa-1), enterococcal surface proteins (esp), cytolysin (cylA, cylL, cylM), gelatinase (gelE), E. faecium endocarditis antigen (EfaA( fm )), hyaluronidase (hyl), and collagen adhesion (Ace) was assessed. Genetic relatedness was determined by pulsed-field gel electrophoresis (PFGE). Twenty-nine E. faecium isolates were obtained and the majority of isolates (n/N = 22/29) were from stool specimens. PFGE analysis identified eight pulsotypes (A-H) based on 80 % similarities. Isolates were represented in five major pulsotypes: type A (n = 5), type B (n = 3), type D (n = 6), type E (n = 5), and type F (n = 7). All isolates were vanA gene-positive. Thirteen isolates had vanA(+)/vanB(+) genotype. Of these, ten exhibited a vanB phenotype and three had a vanA phenotype. Eight isolates with vanA(+)/vanB(-) genotype exhibited vanB phenotype. Six of these eight isolates belonged to the same pulsotype. All isolates were positive for gelE, esp, and EfaA( fm ) genes. Five were CylA-positive and 24 had the hyl genes. Of the eight isolates harboring a combination of gelE, esp, EfaA( fm ), and hyl genes, five showed vanB phenotype-vanA genotype incongruence. This is the first report of vanB phenotype-vanA genotype incongruent E. faecium in the Middle East region. Molecular typing indicates clonal spread and high occurrence of virulence genes, especially esp genes, associated with epidemic clones.

Objectives: Patients with chronic hepatitis B virus (HBV) may exhibit significant liver pathology despite alanine aminotransferase (ALT) and HBV DNA levels below the cutoff values advised by treatment guidelines. We evaluated candidacy for HBV therapy when baseline histopathological changes are taken into consideration. Methods: Clinical, biochemical, serological, virological, and histopathological (METAVIR score) data of 117 patients with HBeAg-negative chronic HBV genotype D were collected and analyzed. Results: Significant pathology (≥F2 and/or ≥A2) and fibrosis (≥F2 ± ≥A2) were found in 73 (62.4%) and 59 (50.4%) patients, respectively. Based on HBV DNA (>2,000 IU/ml) and ALT levels >2 × 40 U/l (the standard cutoff value), only 31 (26.5%) patients were candidates for therapy. This increased to 58 (49.6%) patients when the new ALT cutoff values (30 U/l for males, and 19 U/l for females) were applied. Relying on either ≥F2 and/or A ≥2 or ≥F2 ± ≥A2 increases the treatment candidacy to 73 (62.4%) and 59 (50.4%) patients, respectively. Also, when compared with standard ALT cutoff values, applying both new ALT cutoff values with either significant pathology or fibrosis increases treatment candidacy to 28 (23.9%) and 42 (35.9%) patients, respectively. Conclusion: Liver pathology is more reliable than ALT and HBV DNA in the decision to treat patients with HBeAg-negative chronic HBV genotype D.

Background/Aim Kinesin family member 1B (KIF1B) gene resides in the chromosomal region 1p36.22 and has been reported to have frequent deletions in a variety of human cancers. A recent genome wide association study (GWAS) study conducted on a Chinese population has reported the involvement of a KIF1B genetic variant in Hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). This study aims to investigate the significance of KIF1B genetic variations in HBV-associated hepatitis in patients of Saudi Arabian ethnicity. Methods TaqMan genotyping assay was used to investigate the association of three SNPs (rs17401966, rs12734551, and rs3748578) in 584 normal healthy controls and 660 HBV-infected patients. The patients were categorized into inactive carriers (Case I), active carriers (Case II), Cirrhosis (Case III) and Cirrhosis-HCC (Case IV) sub-groups. Results Since SNPs rs12734551 and rs3748578 are in strong linkage disequilibrium (LD) with rs17401966, only results for the latter SNP are reported. Therefore, the allele frequency of rs17401966 among HBV-infected patients and healthy controls were comparable and therefore, no significant association was observed (P = 0.2811, Odds Ratio (OR) 0.897). A similar analysis was performed among the different sub-groups in order to determine whether KIF1B SNPs were associated with the advancement of the disease. No significant differences were observed in any of the comparisons performed. Conclusion Polymorphisms at KIF1B gene locus investigated in this study showed no significant association with HBV infection or with HBV-associated diseases such as liver cirrhosis or HCC.

[35S]Methionine-labelled envelope polypeptides of herpes simplex virus type 1, strain F, propagated in mammalian cell culture of various origins, were separated by ion-exchange high-performance liquid chromatography on a TSK DEAE-3SW column. Analysis of the fractions by radioimmunoprecipitation followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis of the immunoprecipitates showed similarities as well as distinct differences in the number, migration patterns and molecular mass of the synthesized polypeptides, depending on the host cell. The results show that this method can be used to demonstrate species-specific or organ-specific differences in the processing of virus-specified polypeptides synthesized in host cells.

The purpose of this investigation was to describe the first documented carbapenem-resistant Klebsiella pneumoniae (CRKP) outbreak in a tertiary care facility in Saudi Arabia. We initiated a prospective study to follow all cases of CRKP as well as the active surveillance of patients in areas where cases were identified. We also conducted a retrospective review of the microbiology database for any missed cases of CRKP. Pulsed field gel electrophoresis (PFGE) was conducted for the available CRKP isolates. During March 2010, a cluster of eight CRKPs was detected primarily in the adult intensive care unit (ICU). Patients with CRKPs were put under strict contact isolation, along with appropriate infection control measures. A retrospective review of K. pneumoniae isolates over the previous 6 months revealed two more CRKPs. The PFGE results during the outbreak period showed that the majority of strains were genetically indistinguishable or closely related. The majority of patients had prolonged hospital stay (91%), indwelling devices (81%), surgical procedures (74%), carbapenem use (62%), and colonization/infection with other multiple drug-resistant organisms (MDROs) (57%). Two-fifths of patients with CRKP had clinical infection and 38% died during the current hospitalization. Contact isolation, hand hygiene, environmental cleaning, and staff education may control CRKP outbreak in the acute care setting, but did not prevent endemicity.

The in vitro activity of cefepime, a new aminothiazolyl methoxyamino cephem, was compared with other commonly used antimicrobial agents against 425 strains of Enterobacteriaceae, 138 isolates of other Gram-negative bacilli and 334 strains of Gram-positive cocci. The minimum inhibitory concentrations (MICs) of cefepime were between < 0.03 and 0.5 mg/L for 90% of the Enterobacteriaceae isolates tested, and all 60 isolates of Enterobacter cloacae and Serratia marcescens were inhibited by < 0.03 to 4.0 mg/L of this cephem. Cefepime was the most effective of the drugs tested against Aeromonas hydrophila; its activity against Pseudomonas aeruginosa, Xanthomonas maltophilia and Acinetobacter was comparable to that of ceftazidime and ceftriaxone. All the isolates of methicillin-susceptible Staphylococcus aureus were inhibited by a concentration of cefepime 0.5 to 8.0 mg/L. Cefepime inhibited all 176 isolates of Streptococcus pyogenes, S. agalactiae and S. pneumoniae at concentrations of < 0.015 to 1.0 mg/L. Like other members of its class, it had little or no activity against Xanthomonas, methicillin-resistant S. aureus and enterococci.

Earlier work Tayeb et al. [Tayeb et al. (2008): J Med Virol 80: 1919-1929] set out to study the epidemiology of diarrhea viruses in pediatric populations. The study addressed initially rotavirus, enteric adenovirus, and astrovirus but was later expanded to include norovirus (NoV). Viruses were sought in fecal specimens and characterized for genotype using molecular methods (PCR, RT-PCR, and RFLP) for the first time in KSA. The survey focused on three locations; Jeddah, Makkah, and Riyadh. During the Hajj, the chief population fluxes are via Jeddah to Makkah. One thousand samples were obtained from children (aged 6 years or less) presenting with diarrhea and thus representing community acquired rather than nosocomial infections. Rotavirus was identified in 6% of the samples followed by NoV accounted for 3.5%, astrovirus 1.9%, and adenovirus 1.4%. Rotavirus G9 was characterized for the first time in Saudi Arabia. Adenoviruses were confirmed and further typed using hexon-specific PCR and RFLP. These data were published by Tayeb et al. [Tayeb et al. (2008): J Med Virol 80: 1919-1929]. However, the nature of astrovirus identified was not investigated further. Therefore, more analysis details are appropriate for astrovirus in the Kingdom. As an extension of earlier work carried out on astrovirus serotype distribution in the Kingdom [Tayeb et al. (2008): J Med Virol 80: 1919-1929], a major objective of the project is to use molecular methods to determine the distribution of astrovirus genotype. Such data will help to provide valuable insights into genetic identities and possible sources of virus strains involved not only in pediatric gastroenteritis but also possible outbreaks in the community.

The emergence of chloroquine resistance in Plasmodium falciparum is a significant public health problem where malaria is endemic. We aimed to evaluate the efficacy of pyrosequencing to assess chloroquine resistance among P. falciparum isolates from the southwestern region of Saudi Arabia by analyzing the K76T and N86Y mutations in the P. falciparum chloroquine resistance transporter (PfCRT) and P. falciparum multidrug resistance 1 (PfMDR1) genes, respectively. Blood samples (n = 121) from microscopically positive P. falciparum cases were collected. DNA was extracted, and fragments from each of the genes were amplified by PCR using new sets of primers. The amplicons were sequenced using a pyrosequencer. All of the 121 samples were amplified for assessment of the PfCRT K76T and PfMDR1 N86Y mutations. All of the samples amplified for the PfCRT 76T mutation harbored the ACA codon (121/121; 100%), indicating the presence of the 76T mutation. For the PfMDR1 N86Y mutation, 72/121 samples (59.5%) had the sequence AAT at that position, indicating the presence of the wild-type allele (86N). However, 49/121 samples (40.5%) had a TAT codon, indicating the mutant allele (Y) at position 86. This study shows that pyrosequencing could be useful as a high throughput, rapid, and sensitive assay for the detection of specific single nucleotide polymorphisms in drug-resistant P. falciparum strains. This will help health authorities in malaria-endemic regions to adopt new malaria control strategies that will be applicable for diagnostic and drug resistance assays for malaria and other life-threatening pathogens that are endemic in their respective countries.

We modified the conventional PFGE procedure for Serratia marcescens to establish a rapid method. Our protocol showed modification in the bacterial lysis, washing, and restriction enzyme digestion time. This resulted in shortening the time needed by about 3 days compared to the conventional PFGE method.

Introns from the Epstein–Barr virus (EBV) BART RNAs produce up to 20 micro RNAs (miRNAs) but the spliced exons of the BART RNAs have also been investigated as possible mRNAs, with the potential to express the RPMS1 and A73 proteins. Recombinant RPMS1 and A73 proteins were expressed in Escherichia coli and used to make new monoclonal antibodies that reacted specifically with artificially expressed RPMS1 and A73. These antibodies did not detect endogenous expression of A73 and RPMS1 proteins in a panel of EBV-infected cell lines representing the different known types of EBV infection. BART RNA could not be detected on Northern blots of cytoplasmic poly(A)+ RNA from the C666.1 NPC cell line and BART RNA was found to be mainly in the nucleus of C666.1 cells, arguing against an mRNA role for BART RNAs. In contrast, some early lytic cycle EBV mRNAs were found to be expressed in C666.1 cells. Artificially expressed A73 protein was known to be able to bind to the cellular RACK1 protein and has now also been shown to be able to regulate calcium flux, presumably via RACK1. Overall, the results support the conclusion that the miRNAs are functionally important products of BART transcription in the cell lines studied because the A73 and RPMS1 proteins could not be detected in natural EBV infections. However, the possibility remains that A73 and RPMS1 might be expressed in some situations because of the clear potential relevance of their biochemical functions.

The first straightforward lipase-catalyzed enantioselective access to enantiomerically enriched tiaprofenic acid as a versatile method in chiral separation of racemates is demonstrated. The latter was directly monitored by enantioselective HPLC using a 3,5-dimethylphenylcarbamate derivative of cellulose-based chiral stationary phase namely Chiralpak IB (the immobilized version of Chiralcel OD). Non-standard HPLC organic solvents were used as diluent to dissolve the "difficult to dissolve" enzyme substrate (the acid) and as eluent for the simultaneous enantioselective HPLC baseline separation of both substrate and product in one run without any further derivatization. The existence of a non-standard HPLC organic solvent (e.g., methyl tert-butyl ether) in the mobile phase composition is mandatory to accomplish the simultaneous enantioselective HPLC baseline separation of both substrate and product.

A screening study was conducted to examine the effect of a series of synthesized pyrazoloquinoline derivatives on the growth of Leishmania donovani promastigotes. Sixteen compounds were tested, ten of which showed an inhibitory effect on the growth of promastigotes. Compound 1 demonstrated potent antileishmanial activity, followed by compounds 3 and 7. Some compounds showed less significant activities, while others exhibited little or no activity. Some of these compounds may be potential candidates for future treatment of leishmaniasis.

Survivin, an inhibitor of apoptosis, is over-expressed in foetal tissues and human cancers, but it is almost undetectable in normal tissues. Here we have assessed the level of the survivin protein in some benign tumors of the nervous system: meningioma, schwannoma, low-grade ependymoma, pilocytic astrocytoma and pituitary adenoma. Using immuno-blot analysis we present evidence that these low-grade tumors are positive for survivin expression. In agreement, flow cytometrical analysis showed that both spontaneous and radiation-induced apoptosis levels are very low in these neoplasms. Using host cell reactivation assay we have also shown that these tumor cells are proficient in the repair of gamma-ray-induced DNA damage. However, they are deficient in the removal of ultraviolet (UV) light-induced DNA photolesions, especially the shwannoma- and the pituitary adenoma-derived cells. These results suggest that survivin overexpression may be an early event in the stepwise tumoregenesis and hence could be responsible for the onset as well as the growth advantage during tumoregenic progression of malignant as well as benign neoplasms.

MTS, a tetrazolium dye, is reduced by hydrogenases in living cells to a water-soluble formazan. When it is added to the medium at the end of a cytopathic effects (CPE) inhibition interferon assay, the formazan formed diffuses into the medium; the resultant optical density directly and quantitatively measures how much cellular damage has been produced by the challenge virus in the presence of different amounts of interferon. The use of MTS has considerable advantages in that after it is added, no further steps, such as washing of the cells, extraction of dye, or other manipulations, are needed.

This study was conducted to examine the sensitivity of primary skin fibroblasts from Saudi thyroid cancer (TC) patients to ultraviolet (UV) irradiation. Cell survival was studied by a colony forming assay and DNA repair defects with a host cell reactivation (HCR) assay using UV-irradiated Herpes Simplex Virus (HSV). In addition, p53 gene expression was examined in the same TC cells exhibiting enhanced radiosensitivity. Skin fibroblasts from TC patients (n=4) showed significantly enhanced sensitivity to UV radiation. The average UV dose to reduce survival to 37% of the initial survival (D(37)) value (in Jm(-2)) for fibroblasts from TC patients was 4.6 (3.7-5.6) compared to 7.3 (6.3-8.3) for healthy individuals (n=3). UV-sensitive xeroderma pigmentosum (XP) cells, which were used as positive control, were found to be extremely sensitive with a D(37) value of 0.6 Jm(-2). In a host cell reactivation assay, UV-irradiated HSV was tested for its plaque-forming ability (PFA), by plating infected fibroblasts from TC patients (used as host cells) on African Green Monkey (Vero) kidney cells to form plaques. A significant reduction in the PFA of the UV-irradiated virus (about three fold) on TC cells compared to fibroblasts from the healthy subjects was seen, suggesting a DNA-repair deficiency in the primary fibroblasts of the TC patients. Furthermore, no significant accumulation in radiation-induced p53 expression was observed in cells from the TC patients. Our results, based on a relatively small group of subjects, indicate that Saudi TC patients primary fibroblasts (non-cancerous in nature) may be carriers of cancer-susceptible gene(s) arising from defective DNA repair/processing. These results warrant a larger study to investigate the role of UV-induced bulky DNA damage in thyroid cancer susceptibility.

Synthesis of a novel series of structurally related pyrazoloquinoline nucleosides is described. All the newly synthesized compounds were examined for their in vitro antiviral activity against herpes simplex type-1 as shown by two different bioassays, namely; crystal violet staining or the MTS tetrazolium dye measurement. The acute toxicity (LD50) values of the biologically active compounds were determined.

To evaluate and compare 3 widely used molecular techniques, namely, restriction endonuclease analysis of plasmid deoxyribonucleic acid (REAP), randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) for their suitability and usefulness in the typing and fingerprinting of bacterial isolates.Twenty-four epidemiologically unrelated methicillin-resistant Staphylococcus aureus (MRSA) isolates were used to evaluate the molecular typing methods (REAP, RAPD and PFGE). The study was conducted at the Research and Diagnostic Laboratories of King Faisal Specialist Hospital and Research Center from January 2002 through January 2003.Only 20.8% of all isolates studied were of the same genotypes by all 3 methods. Two major clusters of strains each representing 33% of the total number of isolates were identified by REAP analysis. Each of RAPD and PFGE however, identified one major cluster represented by 54% and 83% of the total number of isolates, all 3 typing methods, therefore, showed the clonal genetic relatedness among distant MRSA isolates. However inter-strain comparison of fingerprint data generated from each method revealed differences in clonal representation of the MRSA isolates.Although a variety of molecular assays are available for typing of bacterial species, there is no single standardized protocol for routine analysis. Reproducibility and interpretation of genotypic data are therefore, highly dependent on methodologies employed by the individual laboratory. Our findings illustrate the importance of using a combination of methods in typing schemes of bacterial isolates. In terms of reproducibility and typeability we found that PFGE is superior to REAP and RAPD and, therefore, more suitable for routine, standardized tracing of nosocomial bacterial isolates.

Background: Sparfloxacin is a new fluoroquinolone with excellent in vitro activity against a variety of Gram-positive and Gram-negative bacteria. Methods: In vitro antibacterial activity of this new drug was tested against 139 strains of Brucella melitensis. These strains were isolated from 138 patients at a major tertiary care referral center in Riyadh, Saudi Arabia. Results: Seven percent of the strains were inhibited by 0.06 mg per L and 99% by 0.12 mg per L of sparfloxacin. Of the five other fluoroquinolones tested, ciprofloxacin, norfloxacin, pefloxacin, and fleroxacin exhibited comparable or slightly higher minimal inhibitory concentrations values. They inhibited the growth of 138 of the 139 isolates at 0.25-0.5 mg per L. Rufloxacin required 2-8 mg per L to inhibit 138 of the 139 strains tested. One of the isolates, which had previously developed resistance to ciprofloxacin during therapy with this drug, with a rise in MIC from 0.06 mg per L to >5.0 mg per L, exhibited cross-resistance to sparfloxacin and other fluoroquinolones. Conclusion: Sparfloxacin exhibited excellent in vitro activity against clinical isolates of Brucella. Clinical trials are warranted to determine its potential in the treatment of brucellosis.

The transient control of diverse biological responses that occurs in response to varied forms of stress is often a highly regulated process. During the interferon (IFN) response, translational repression due to phosphorylation of eukaryotic initiation factor 2alpha, eIF2alpha, by the double-stranded RNA-dependent protein kinase, PKR, constitutes a means of inhibiting viral replication. Here we show that the transient nature of the IFN response against acute viral infections is regulated, at least in part, by RNase L. During the IFN antiviral response in RNase L-null cells, PKR mRNA stability was enhanced, PKR induction was increased, and the phosphorylated form of eIF2alpha appeared with extended kinetics compared with similarly treated wild type cells. An enhanced IFN response in RNase L-null cells was also demonstrated by monitoring inhibition of viral protein synthesis. Furthermore, ectopic expression of RNase L from a plasmid vector prevented the IFN induction of PKR. These results suggest a role for RNase L in the transient control of the IFN response and possibly of other cytokine and stress responses.

Conventional diagnosis of infection with Giardia duodenalis is by faecal examination but the sensitivity of a single examination is low. Serological tests, although not always positive, are more acceptable to patients and are useful in determining the prevalence of giardiasis in large populations. We show here that blood collected on filter paper and dried provides an excellent source of material for enzyme-linked immunosorbent assays; using samples from a group of 88 stool-negative and 45 positive patients, the optimum results were obtained by taking the control mean optical density plus one standard deviation as the negative/positive cut-off value. The sensitivity was 91% ( 2 45 false negatives), and the specificity 95% ( 4 88 false positives). This method should be particularly useful for large-scale surveys in developing countries or wherever serological testing is done in central laboratories.

BackgroundHepatitis C virus (HCV) shows remarkable genetic diver-sity, which contributes to its high persistence and variedsusceptibilities to antiviral treatments. Previous studieshave reported that the substitution of amino acids in theHCV-1b core region at positions 70 (Arg70 to Gln70)and/or 91 (Lue91 to Met91) is associated with a poorresponse to pegylated- interferon and ribavirin (PEG-IFN/RBV) therapy [1,2]. Because the role of the core protein inHCV infections is unclear in Saudi populations, we aimedin this study to analyze the full-length core proteinsequences from Saudi patients.Materials and methodsA total of 300 samples were obtained from Saudi patientswho went through PEG-IFN/RBV treatment. Sampleswere divided further to responder and non-respondergroups. Direct sequencing was employed, followed bysequence analyses using advanced software.ResultsOurdatashowedthattherewassignificantassociationbetween core protein mutations, particularly at position70, and treatment outcome in HCV1b and HCV-4d butnot in HCV-1a and HCV-4a clinical samples. In addition,amino acid residue at position 91 was well-conservedamong all clinical samples where Cys91 is the dominantamino acid residue. Furthermore, our data reported pointmutations at different positions that were flagged as ‘rare’mutations by negative BLOSUM scores.ConclusionsAmino acid substitution pattern differs substantiallyamong HCV sub-genotypes. Such discrepancy needsfurther investigations. Our finding provides a new insightinto HCV among effected Saudi population where theknowledge of HCV polymorphisms is lacking.

The 5′-untranslated region (5′-UTR) sequences of 33 GB virus C/hepatitis G virus (GBV-C/HGV) obtained from different geographic areas were determined through reverse-transcription polymerase chain reaction and dideoxy chain termination sequencing, the alignment of sequences, the estimation of the number of nucleotide substitution per site, and construction of phylogenetic trees. The 5′-UTR of GBV-HGV was found to be heterogeneous, with 70.9–99.5% homology. Three distinct phylogenetic branches were observed consistently in all phylogenetic trees. GBV-C is the prototype for one, HGV for another, and there is a new branch which consisted of GBV-C/HGV isolates from Asia. Genotype-specific restriction sites for the restriction enzymes, ScrFI and BsmFI, were identified, and a simple restriction fragment polymorphism analysis was developed for genotyping. These data provide evidence that GBV-C/HGV consists of three different genotypes. Our simple genotyping assay will also provide a tool for epidemiological studies of GBV-C/HGV infection.

Sera from 164 Saudi Arabian patients with non-A, non-B hepatitis liver disease were examined for antibodies to hepatitis C virus (HCV) by second- and third-generation recombinant immunoblot assay (RIBA-2 and RIBA-3) and for HCV RNA by polymerase chain reaction (PCR). By using RIBA-2, 92 (56.1%) were reactive, 64 (39%) were nonreactive, and 8 (4.9%) were indeterminate. By using RIBA-3, 98 (59.7%) were reactive 60 (36.6%) were nonreactive, and 6 (3.7%) were indeterminate. By using PCR, 108 (65.9%) were positive. Of the eight RIBA-2 indeterminate samples, seven became RIBA-3 reactive but PCR-positive, and one became RIBA-3 nonreactive but PCR-negative. Of the six RIBA-3 indeterminate samples, five were RIBA-2 nonreactive but PCR-positive, and one was RIBA-2 reactive but PCR-negative. From our study on Saudi patients, we conclude that RIBA-3 has slightly but not significantly improved the results of anti-HCV antibody detection, and is probably of more value to resolve those indeterminate samples by RIBA-2. Although expensive, PCR remains the most reliable HCV diagnostic method until an HCV antigen detection test is available.

The JC polyomavirus (JCV) is ubiquitous in humans, infecting children asymptomatically, then persisting in renal tissue. Since JCV DNA can be readily isolated from urine, it should be a useful tool with which to study the evolution of DNA viruses in humans. We showed that JCV DNA from the urine of Japanese, Taiwanese, Dutch and German patients can be classified into A and B types, based upon restriction fragment length polymorphisms (RFLPs). This work was extended in the present study. We established multiple JCV DNA clones from the UK, Spain, Italy, Sweden, South Korea, People's Republic of China, Malaysia, Indonesia, Mongolia, India, Sri Lanka, Saudi Arabia, Ethiopia, Kenya, Zambia, South Africa and Ghana. Using type-specific RFLPs, most clones except the four clones from Ghana were classified as either type A or B. We constructed a molecular phylogenetic tree for the Ghanaian clones and several representative type A and B clones. According to the phylogenetic tree, the Ghanaian clones constituted a major new group, tentatively named type C. From the findings presented here and elsewhere, the following conclusions were drawn: (i) type A is prevalent only in Europe; (ii) type B is found mainly in Asia and Africa; and (iii) type C is localized to part of Africa. Our findings should help to clarify how JCV evolved in humans.

We describe our experience in detecting and containing the first documented CRKP outbreak in Saudi Arabia (SA).

Twelve samples of cerebrospinal fluid (CSF) from Saudi pediatric patients with suspected encephalitis were found negative for herpes simplex virus by cell culture and by nucleic acid hybridization using(32)P-labeled specific probes. After amplification of DNA extracted from 100 microl of each sample using primers for the glycoprotein D gene of HSV, seven specimens were found positive for HSV DNA. Subsequent hybridization with the labeled DNA probe ensured the positivity of the seven samples and further detected one more positive sample, for a total of eight HSV DNA positive cases. This technique improved the detection of HSV in CSF samples and may assist in an early diagnosis for HSV ecephalitis.

Fifty-two clinical isolates of herpes simplex virus type 1 (HSV-1) from Saudi Arabian patients were analysed by restriction endonuclease digestion of the virus DNA using the enzymes Hindlll and BarnHI, followed by hybridization with 32P labelled DNA of laboratory strain F. Of the isolates, 17 were resolved into four distinct cleavage patterns with HindIII restriction enzyme. The remaining 35 strains had the same cleavage pattern as the standard HSV-1-F. Further investigation of the 52 isolates with BarnHI, which is a multicut enzyme and therefore capable of higher resolution, differentiated 47 of the 52 isolates and were assigned into nine cleavage groups. Comparing our findings with similar studies reported elsewhere suggest geographic clustering of HSV-1 strains. Fragments giving rise to the observed DNA polymorphism were mapped to the unique region of the long and short segments of the genome. © 1992 Wiley-Liss, Inc.

High performance liquid chromatography (HPLC) with a TSK-4000SW gel filtration column was used to compare envelope polypeptides from four strains of herpes simplex virus type 1 (HSV-1). The chromatographic profiles demonstrated polypeptide variability among three clinical strains and the wild-type F strain. Radioimmunoprecipitation of the HPLC fractions with polyclonal anti-HSV-1 followed by SDS-polyacrylamide gel electrophoresis (PAGE) of the immunoprecipitates revealed molecular weight differences of various polypeptides in fractions from the area containing major peaks. This HPLC method could prove useful for the analysis of polypeptide polymorphism in clinical isolates of HSV-1, as well as in other viruses.

Sendai virus glycoproteins HN and F were purified by immunoaffinity chromatography from virions disrupted by β- d -octylglucoside . The purified glycoproteins were reconstituted in recombinant vesicles with phosphatidylcholine or phosphatidylethanolamine and phosphatidylserine. P815 or EL-4 cells treated with glycoprotein HN/F-phosphatidylcholine recombinant vesicles acquired the glycoproteins and retained them in the plasma membrane for 4 h as demonstrated by surface immunofluorescence specific for each protein. Cells treated with glycoprotein HN-phosphatidylcholine recombinant vesicles initially bore glycoprotein HN on the surface but the protein eluted within 2 h. Surfaces of cells treated with glycoprotein F-phosphatidylcholine recombinant vesicles did not acquire the glycoprotein. Cells treated with glycoprotein HN-phosphatidylethanolamine: phosphatidylserine recombinant vesicles or glycoprotein F-phosphatidylethanolamine: phosphatidylserine recombinant vesicles in the presence of 5 mM Ca2+ acquired each protein for at least 2 h. Experiments showed that the acquired glycoproteins capped with antibody and that when glycoproteins HN and F were together on the surface they co-capped. Acquired viral glycoproteins did not co-cap with intrinsic H-2 glycoproteins.

Clinical specimens from 159 patients suspected with herpes simplex virus (HSV) were examined by monoclonal antibody immunofluorescence (IF) and by a commercial biotinylated DNA probe kit following cell culture isolation. Herpes simplex virus was isolated from 57 samples. All cultures were positive by IF when the cytopathic effect (CPE) was less than 1+ but only 49 (86%) yielded positive reaction with the DNA probe when CPE was at least 1+. A total of 54 clinical specimens was also examined directly by immunoperoxidase histopathology (IHP), IF, and DNA hybridization. Of these, 16 were positive by IHP, 15 by IF, and only five by DNA probe. The DNA probe kit was found to be reasonably sensitive only after cell culture isolation of HSV. Compared to the IF procedure, the DNA probe kit was found to be costly, labor intensive, and time consuming.

Rabbit kidney (RK-13) and human jejunum and ileum (I-407) cells infected with herpes simplex virus type 1, strain F, were radiolabelled with [14C]glucosamine or [35S]methionine for 24 h. The cells were extracted with 1% Triton X-100 and the extracts were separated by gel filtration high performance liquid chromatography. Monoclonal antibody immunoprecipitation of the fractions collected from the column revealed a monomeric glycoprotein D (gD) of 52 - 56,000 molecular weight from RK-13 cells and two monomeric forms of gD, 54,000 and 58,000 molecular weight, from I-407 cells. Densitometry scanning of the autoradiograms from SDS-PAGE showed gD from the RK-13 host cells to be 98.7% pure with the [35S]methionine label and 97.0% pure with the [14C]glucosamine. On the other hand, gD from the I-407 host cells was only 78.6% with the [35S]methionine label and 96% pure with the [14C]glucosamine. This method could provide a means for the isolation of native gD for structural and immunological studies.

Leishmania donovani, the causative agent of visceral leishmaniasis, is transmitted by sand flies and replicates intracellularly in their mammalian host cells. The emergence of drug-resistant strains has hampered efforts to control the spread of the disease worldwide. Forty-four 1,3,4-thiadiazole derivatives and related compounds were tested in vitro for possible anti-leishmanial activity against the promastigotes of L. donovani. Micromolar concentrations of these agents were used to study the inhibition of multiplication of L. donovani promastigotes. Seven compounds were identified with potential antigrowth agents of the parasite. Compound 4a was the most active at 50μM followed by compound 3a. These compounds could prove useful as a future alternative for the control of visceral leishmaniasis.

A chloroform extract of Khat (Catha edulis) leaves was used to study the cytotoxic activity on KB, 1BR.3, and XP2Bi cells. Log phase cell survival curves showed an LD50 of 40 ng/ml for KB cells. 1BR.3 and XP2Bi cells were biphasic in their response to the extract during log phase, with an LD50 of 20 and 75 ng/ml, respectively. Stationary phase cells were unaffected by the extract. DNA and RNA synthesis inhibition was studied using radiolabeled thymidine or uridine to measure the amount of extract that inhibits the synthesis to 50% of the untreated control cells. DNA synthesis was inhibited by 45, 60 and 200 ng/ml and RNA synthesis by 24, 17 and 58 ng/ml in 1BR.3, XP2Bi and KB cells, respectively. Protein synthesis was inhibited to 15–20% of untreated control cells by a dose of 40 ng/ml in all the cells studied. From this work, it is apparent that the main cause of cytotoxicity of Khat extract may be the inhibition of de novo RNA synthesis. Our results suggest that this effect is exerted on all cells used in this study and that KB cells demonstrate a higher resistance to the toxic component.

Interleukin-8 (IL-8), a proinflammatory chemokine, is induced by viruses and appears in circulation during viral infections. We found that IL-8 enhanced cytopathic effect induced by the positive strand RNA virus, encephalomyocarditis virus (EMCV), in the human WISH cell line. The enhancement was dependent on IL-8 dose and virus dose and was reversible by specific monoclonal antibodies to IL-8. The chemokine was also able to increase EMC viral RNA synthesis and infectious virus yield. This IL-8 enhancing action was not observed in the case of the negative strand RNA virus, vesicular stomatitis virus (VSV), in WISH cells. We examined the activity of constitutive 2′,5′-oligoadenylate synthetase (OAS), a pathway that was implicated in protection from EMCV but not VSV. The IL-8 action in EMCV-infected cells, unlike VSV-infected cells, was associated with decreased OAS activity in a manner that was independent of OAS gene expression. Understanding mechanisms of cytokine enhancement of viral activity may lead to novel ways to control viral infections.

In this work, a direct enantioselective HPLC monitoring system is developed. The system consists of an HPLC equipped with an immobilized cellulose-based chiral stationary phase Chiralpak IB, UV and a polarimetric detector in series. The developed system is used in the direct monitoring of the enantioselectivity of lipase-catalyzed enantioselective esterification of 2-phenoxy propionic acid of which the (R)-enantiomer is a precursor of the (R)-2-(p-hydroxyphenoxy)propionic acid known as an important intermediate in synthesizing aryloxyphenoxypropionic acid based agrochemicals. The versatility of the biocatalyzed reaction in chiral separation to access to both enantiomers of racemic 2-phenoxy propionic acid is demonstrated. The solvent versatility of the immobilized chiral stationary phase used in the direct monitoring of such reaction in non-standard HPLC solvent is shown.