Roy A Khalaf

Lebanese American University, Beyrouth, Beyrouth, Lebanon

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Publications (16)26.45 Total impact

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    ABSTRACT: As leading opportunistic fungal pathogens identification and subtyping of Candida species are crucial in recognizing outbreaks of infection, recognizing particularly virulent strains, and detecting the emergence of drug resistant strains. In this study our objective was to compare identification of Candida albicans by the hospitals through the use of conventional versus identification based on the ITS (Internal Transcribed Spacer) and to assess biofilm forming capabilities, drug resistance patterns and correlate these with MLST typing. ITS typing revealed a 21.2% hospital misidentification rate. Multidrug resistance to three drugs out of four tested was detected within 25% of the isolates raising concerns about the followed treatment regimens. Drug resistant strains as well as biofilm formers were phylogenetically related, with some isolates with significant biofilm forming capabilities being correlated to those that were multidrug resistant. Such isolates were grouped closely together in a neighbor-joining tree generated by MLST typing indicating phylogenetic relatedness, microevolution, or recurrent infection. In conclusion, this pilot study gives much needed insight concerning C. albicans isolates circulating in Lebanese hospitals and is the first study of its kind correlating biofilm formation, antifungal resistance, and evolutionary relatedness.
    BioMed Research International 01/2014; 2014:931372. · 2.88 Impact Factor
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    ABSTRACT: AIMS: The Natural Killer Cell Immunoglobulin-like Receptors (KIR) Genotypes profiling in Follicular Lymphoma has not been reported before in the literature. MATERIALS AND METHODS: DNA extracted from 20 Follicular Lymphoma patients and 62 healthy controls was analyzed for KIR genotyping using a polymerase chain reaction / sequence specific primers technique (PCR/SSP) for the presence of 16 KIR genes and pseudogenes loci. RESULTS: The AA, AB, and BB genotypes frequencies were, respectively, 20%, 60% and 20% with an A:B ratio of 1:1. KIR 2DL4, KIR 3DL2, KIR 3DL3, and KIR 3DP1*003 were presented in all individuals. No significant difference between patients and controls was detected. CONCLUSION: KIR genotyping profile does not seem to be associated with Follicular Lymphoma. The results presented in this pilot research represent the first international report about this important clinical entity.
    Gene 05/2013; · 2.20 Impact Factor
  • Samer Younes, Roy Khalaf
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    ABSTRACT: The opportunistic fungal pathogen Candida albicans is one of the leading agents of life threatening infections affecting immunocompromised individuals. Many factors make C. albicans a successful pathogen. These include the ability to switch between yeast and invasive hyphal morphologies in addition to an arsenal of cell wall virulence factors such as lipases, proteases, dismutases and adhesins that promote the attachment to host, a prerequisite for invasive growth. We have previously characterized Hwp2 a C. albicans cell wall protein which we found necessary for proper oxidative stress, biofilm formation and adhesion to host cells. Baker's yeast Saccharomyces cerevisiae also possesses adhesins that promote aggregation and flocculence. Flo11 is one such adhesin that has sequence similarity to Hwp2. Here we determined that transforming an HWP2 cassette can complement the lack of filamentation of an S. cerevisiae flo11 null strain, and impart on S. cerevisiae adhesive properties similar to a pathogen.
    Microbiology 04/2013; · 3.06 Impact Factor
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    ABSTRACT: Candida albicans is a common opportunistic pathogen that causes a wide variety of diseases in a human immunocompromised host leading to death. In a pathogen, cell wall proteins are important for stability as well as for acting as antigenic determinants and virulence factors. Pir32 is a cell wall protein and member of the Pir protein family previously shown to be upregulated in response to macrophage contact and whose other member, Pir1, was found to be necessary for cell wall rigidity. The purpose of this study is to characterize Pir32 by generating a homozygous null strain and comparing the phenotype of the null with that of the wild-type parental strain as far as filamentation, virulence in a mouse model of disseminated candidiasis, resistance to oxidative stress and cell wall disrupting agents, in addition to adhesion, biofilm capacities, and cell wall chitin content. Our mutant was shown to be hyperfilamentous, resistant to sodium dodecyl sulfate, hydrogen peroxide, sodium chloride, and more virulent in a mouse model when compared to the wild type. These results were unexpected, considering that most cell wall mutations weaken the wall and render it more susceptible to external stress factors and suggests the possibility of a cell surface compensatory mechanism. As such, we measured cell wall chitin deposition and found a twofold increase in the mutant, possibly explaining the above-observed phenotypes.
    Mycopathologia 03/2012; 174(2):107-19. · 1.65 Impact Factor
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    ABSTRACT: The fungal pathogen Candida albicans is a leading causative agent of death in immunocompromised individuals. Many factors have been implicated in virulence including filamentation-inducing transcription factors, adhesins, lipases and proteases. Many of these factors are glycosylphosphatidylinositol-anchored cell surface antigenic determinant proteins. Pga1 is one such protein shown to be upregulated during cell wall regeneration. The purpose of this study was to characterise the role Pga1 plays by creating a homozygous pga1 null strain and comparing the phenotype with the parental strain. It was observed that the mutant strain showed less oxidative stress tolerance and an increased susceptibility to calcofluor white, a cell surface disrupting agent that inhibits chitin fibre assembly which translated as a 40% decrease in cell wall chitin content. Furthermore, the mutant exhibited a 50% reduction in adhesion and a 33% reduction in biofilm formation compared with the parental strain, which was reflected as a slight reduction in virulence. Our data suggest that Pga1 plays an important role in cell wall rigidity and stability. It was also observed that the pga1 null was over filamentous on both liquid and solid media and exhibited increased resistance to SDS suggesting upregulation of filamentation-inducing genes and cell surface components to partially compensate for the deletion.
    Mycoses 11/2011; 54(6):491-500. · 1.28 Impact Factor
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    ABSTRACT: Candida albicans is an important fungal pathogen of humans that is responsible for the majority of mucosal and systemic candidiasis. The host-pathogen interaction in C. albicans has been the subject of intense investigation as it is the primary step that leads to establishment of infection. Hwp2 is a cell wall GPI-anchored cell wall protein that was previously shown to be necessary for hyphal and invasive growth on solid media. The purpose of the current study is to further characterize the protein as far as its role in oxidative stress, sensitivity to cell wall disrupting agents, adhesion to human epithelial and endothelial cells, biofilm formation and chitin content. It appears that Hwp2 is necessary for proper oxidative stress tolerance, adhesion and biofilm formation as an hwp2 null is more susceptible to increasing doses of hydrogen peroxide, unable to adhere efficiently to epithelial and endothelial cell lines and unable to form wild type biofilm levels.
    Microbiological Research 07/2011; 166(5):430-6. · 1.99 Impact Factor
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    ABSTRACT: The fungal pathogen Candida albicans is one of the leading causative agents of death in immunocompromised individuals. It harbors an arsenal of cell wall anchored factors that are implicated in virulence such as filamentation inducing factors, adhesins, lipases, proteases, and superoxide dismutases. Dse1 is a cell wall protein involved in cell wall metabolism. The purpose of this study is to characterize the role Dse1 plays in virulence. Dse1 appears to be an essential gene as no homozygous null mutant was possible. The heterozygote mutant exhibited increased susceptibility to calcofluor white, a cell wall disrupting agent, with a subsequent reduction in cell wall chitin content, decreased oxidative stress tolerance, a 30% reduction in biofilm formation, and a delay in adhesion that was mirrored by a reduction in virulence in a mouse model of infection. Dse1 thus appears to be an important protein involved in cell wall integrity and rigidity.
    Interdisciplinary Perspectives on Infectious Diseases 01/2011; 2011:504280.
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    ABSTRACT: The occurrence of highly conserved amyloid-forming sequences in Candida albicans Als proteins (H. N. Otoo et al., Eukaryot. Cell 7:776-782, 2008) led us to search for similar sequences in other adhesins from C. albicans and Saccharomyces cerevisiae. The beta-aggregation predictor TANGO found highly beta-aggregation-prone sequences in almost all yeast adhesins. These sequences had an unusual amino acid composition: 77% of their residues were beta-branched aliphatic amino acids Ile, Thr, and Val, which is more than 4-fold greater than their prevalence in the S. cerevisiae proteome. High beta-aggregation potential peptides from S. cerevisiae Flo1p and C. albicans Eap1p rapidly formed insoluble amyloids, as determined by Congo red absorbance, thioflavin T fluorescence, and fiber morphology. As examples of the amyloid-forming ability of the native proteins, soluble glycosylphosphatidylinositol (GPI)-less fragments of C. albicans Als5p and S. cerevisiae Muc1p also formed amyloids within a few days under native conditions at nM concentrations. There was also evidence of amyloid formation in vivo: the surfaces of cells expressing wall-bound Als1p, Als5p, Muc1p, or Flo1p were birefringent and bound the fluorescent amyloid-reporting dye thioflavin T. Both of these properties increased upon aggregation of the cells. In addition, amyloid binding dyes strongly inhibited aggregation and flocculation. The results imply that amyloid formation is an intrinsic property of yeast cell adhesion proteins from many gene families and that amyloid formation is an important component of cellular aggregation mediated by these proteins.
    Eukaryotic Cell 03/2010; 9(3):393-404. · 3.59 Impact Factor
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    ABSTRACT: Various factors are thought to be responsible for Candida albicans virulence, such as lipases, proteases and adhesins. Many of these factors are GPI-anchored cell surface proteins responsible for pathogenicity. Hwp2 is a putative GPI-anchored protein. The purpose of this study is to characterize the role of Hwp2 regarding filamentation on various filamentation-inducing and non-inducing solid and liquid media, virulence in a mouse model of disseminated candidiasis, and drug resistance to six widely used antifungal agents, by creating a homozygous null hwp2 strain and comparing it with the parental and a revertant HWP2+strain. It was observed that an hwp2Δ strain was highly filamentation-deficient on solid agar media as opposed to most liquid media tested. Furthermore, the mutant strain was slightly reduced in virulence compared to the wild strain since all mice infected with the control strain died after 6 days of injection compared with 11 days for the mutant. These results indicate a possible role for Hwp2 in adhesion and invasiveness. Finally a previously unidentified 37-amino-acid-long, stretch of Hwp2, possibly involved in protein aggregation, was found to align with high sequence identity and exclusively to C. albicans cell wall proteins.
    Microbiological Research 01/2010; · 1.99 Impact Factor
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    ABSTRACT: Genotypic profiles of the natural killer cell immunoglobulin-like receptors (KIR) have been reported to vary among different ethnic groups and variable clinical entities. This study represents the first report on its distribution among patients with familial Mediterranean fever (FMF). We studied 56 unrelated Lebanese FMF patients, had their DNA typed using sequence-specific primer (SSP) technique for the presence of 16 KIR gene and pseudogene loci, and compared them to the general Lebanese population. The AA1 genotype was the most frequent in both the FMF and control groups. Six new KIR profiles were identified. The FMF group showed a higher prevalence of KIR 3DP1*003 (p<0.05) and an increase in the BB genotype compared with controls. The results lead to an interesting future research question of whether or not KIR genotype is involved in the predisposition to or pathogenesis of FMF. This is the first report that describes the KIR genotypic profile in this important clinical disease.
    Genetic Testing and Molecular Biomarkers 03/2009; 13(1):91-5. · 1.44 Impact Factor
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    ABSTRACT: The incidence of antifungal resistance is on the increase worldwide and novel drugs are constantly being developed to counter this trend. One hundred and sixteen clinical isolates of Candida albicans were collected from Lebanese hospitals in order to first determine the degree of resistance of Lebanese isolates to four common azoles: fluconazole (FL), itraconazole (IT), ketoconazole (KE), and voriconazole (VO), in addition to amphotericin B (AP) and caspofungin (CS) through the Epsilometer test method and second, determine any relationship between the allelic compositions of the mating type loci (MTLa, MTL alpha, MTLa/alpha) with drug resistance. Results showed that resistance, among C. albicans isolates, was the highest with 12% for IT, followed by 7.7% for VO, 6% for KE, 5% for FL, 1.7% for AP and 0% for CS. Three isolates (2.6%) were resistant to all azoles tested, including one that was resistant to all drugs used except CS. Eleven isolates were homozygous at the MTL locus (9.5%), five of which (45%) were resistant to at least one antifungal drug whereas 14 of the 105 heterozygous strains (13%) exhibited similar resistance (P = 0.02), indicating a strong correlation between MTL locus homozygosity and resistance.
    Mycoses 03/2009; 52(2):141-8. · 1.28 Impact Factor
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    ABSTRACT: This study represents the first report on the distribution of killer cell immunoglobulin-like receptor (KIR) genotype among recurrent tonsillitis patients. We recruited 34 Lebanese pediatric patients diagnosed with recurrent tonsillitis and had their DNA typed using sequence-specific primer technique for the presence of 16 KIR loci. We observed that 25 different KIR genotypes were present similar to the general control population with the same KIR gene content. There was no statistically significant difference in the distribution of the activating and inhibitory KIR genes between the two categories. Like in the general control population, we noted a predominance of the AB genotype; however, the KIR genotypic distribution among the tonsillitis patients was much more heterogeneous with even new genotypes not reported in the control group. Although the sample size is small, this first study observes an interesting heterogeneous KIR gene profile in recurrent tonsillitis that warrants larger and further research in the area for the true biological and clinical significance of this observation.
    Genetic Testing 01/2009; 12(4):517-21. · 1.17 Impact Factor
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    ABSTRACT: Candida albicans is a dimorphic pathogenic fungus that causes mucosal and systemic infections. C. albicans pathogenicity is attributed to its ability to exist in different morphologic states and to respond to stress by up regulating several key genes. DDR48 is a stress-associated gene involved in DNA repair and in response to antifungal drug exposure. One allele of DDR48 was knocked out by homologous recombination that inserted a marker cassette in its position. Furthermore, reintroducing DDR48 on a plasmid created a revertant strain. Strains were grown on filamentation inducing and noninducing media, subjected to an oxidative stress challenge, injected into mice to assess virulence, and assayed for antifungal susceptibility by the E-test method. DDR48 was found to be haploid insufficient and possibly essential, since only a heterozygote, but not a homozygous, null mutant was generated. The mutant was filamentation defective on all hyphal media tested including serum and corn meal agar. Discrepancies in drug resistance profiles also were present: compared with the parental strain, DDR48/ddr48 heterozygote strain was susceptible in a dose-dependent manner to itraconazole and fluconazole and susceptible to ketoconazole. The mutant also appeared to be hypersensitive to a potentially lethal hydrogen peroxide challenge. However, no reduction in virulence of the mutant was observed. The present findings provide evidence that DDR48 is essential for filamentation, stress response, and possibly viability of C. albicans, making it a prime target for antifungal drug design.
    Medical science monitor: international medical journal of experimental and clinical research 07/2008; 14(6):BR113-121. · 1.22 Impact Factor
  • Ghyda Barada, Raida Basma, Roy A Khalaf
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    ABSTRACT: The present study involves collecting 125 isolates labeled as C. albicans from five different Lebanese hospitals and utilizing the microsatellite genotyping test to determine the following: first, the accuracy of hospital identification by comparing microsatellite results to hospital results. Second, the frequency and genotypes of infectious strains present relative to tissue and hospital location--a possible indicator of nosocomial infection, and third, a possible relationship between lack of microsatellite heterozygosity to azole resistance. Our results showed that the error in hospital identification varied from 2 to 33%, averaging at 7%, with the highest identification error in stool. Misidentified isolates were mainly Candida tropicalis followed by C. glabrata and C. parapsilosis. Strains with similar genotypes were also found to occur within certain hospitals suggesting the possibility of nosocomial infection. Finally, a relationship between lack of heterozygosity and azole resistance was observed since nine out of 10 homozygous isolates sharing a common allele with a heterozygote strain were sensitive to all drugs tested, whereas the homozygous genotype was resistant to at least one drug.
    Mycopathologia 04/2008; 165(3):115-25. · 1.49 Impact Factor
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    ABSTRACT: The fungus C. albicans is among the leading agents causing death in immunocompromised patients. Most hospitals rely on conventional morphological techniques such as the germ-tube assay and the API system for correct identification. This technique is subjective and hence error prone. Recently, more and more molecular techniques for correct identification have been developed. The latest is the LightCycler real-time PCR technique coupled with melting curve analysis. One hundred hospital isolates presumed to be C. albicans from four major Lebanese hospitals were tested using the real-time PCR technique. The results were compared with a germ-tube test. Furthermore, all real-time PCR-positive samples were replica plated on growth media at 30 degrees C and 45 degrees C to differentiate between C. albicans and C. dubliniensis. Finally, all PCR-negative samples were identified using the API 20C AUX yeast identification system. Twenty-four hospital isolates were non-albicans by PCR (p<0.001). Of these samples, 17 were germ tube positive (p<0.001). A further 6 samples showed positive identification by PCR, but were germ tube negative (p=0.015). Three of the 24 C. albicans-negative isolates were misidentified by the API 20C AUX. None of the C. albicans real-time PCR-positive samples failed to grow at 45 degrees C, the C. dubliniensis non-permissive temperature. Considering the impact of false identification on the general public health through the use of wrong antifungal drug treatment and the emergence of novel drug-resistant strains, hospitals should update their classification methods using molecular techniques.
    Medical science monitor: international medical journal of experimental and clinical research 06/2007; 13(5):MT7-12. · 1.22 Impact Factor
  • Human Immunology - HUM IMMUNOL. 01/2007; 68(1).

Publication Stats

101 Citations
26.45 Total Impact Points

Institutions

  • 2008–2014
    • Lebanese American University
      • Department of Natural Sciences
      Beyrouth, Beyrouth, Lebanon
  • 2010
    • The University of Arizona
      Tucson, Arizona, United States
  • 2009
    • American University of Beirut
      • Department of Pathology and Laboratory Medicine
      Beirut, Mohafazat Beyrouth, Lebanon
  • 2007
    • Case Western Reserve University
      • Department of Genetics and Genome Sciences
      Cleveland, OH, United States