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Thalassemia is best thought of as a group of disorders rather than a single disease. They demonstrate a hemoglobin synthesis disorder in which there exists a defect in the rate of production of one or more of the globin chains. This defect results from either a heterozygous or homozygous deletion or inactivation of a globin chain gene.Thalassemias are named according to the affected gene or globin chain which is showing reduced or absent synthesis. Globin chain gene loci are found on the following chromosome locations:Chromosome 11 (Beta, Delta, Epsilon, and Gamma)Chromosome 16 (Alpha, and Zeta)

Chromosome 16 contains the genetic codes for the zeta and alpha hemoglobin chains.Each chromosome has two loci alpha chains α1 and α2. This equals a total of four gene loci coding for the alpha hemoglobin chain. See the image for a visual representation of these loci.In the genotypic notation of alpha thalassemia an "α" represents the presence of an alpha locus. A "-" represents a deletion of a locus.The notation for the normal number of alpha loci is αα/αα. The amount of Hb A produced by this normal gene is 95-98 %.(drawing modified from Harmening, 1999)

Why our immune system malfunctions is not completely understood. One current hypothesis is that the following series of events occurs resulting in the initiation of an autoimmune reaction. Gender and Genetic PredispositionA predisposition is usually the first step toward the development of an autoimmune reaction. Women are more likely to develop a systemic autoimmune disease than men. For example in SLE the female to male ratio is 9:1. The genotype of some individuals predetermines that their immune system will be more prone to a break in tolerance. This genetic susceptibility appears to be linked to multiple genes rather than a single gene. This is supported by evidence that some autoimmune diseases are more frequently encountered in certain ethnic groups compared to others. For example in American women between the ages of 15 and 64, the prevalence of SLE is 1 in 700 for Caucasians while it is 1 in 245 for African-American women.(Ref1) Evidence in one recent study suggests that the genes that impart an increased resistance to malaria unfortunately produce an increased susceptibility to the systemic autoimmune rheumatic diseases.(Ref2)Triggering eventThe second step is the occurrence of a triggering event that leads to a break in tolerance. For some very susceptible individuals this event might be exposure to an environmental trigger. These environmental triggers could be ubiquitous such as exposure to the Epstein Barr virus (EBV), or very limited, such as the exposure to leaking silicon from a breast implant. In others, the triggering event might be a change in hormonal balance. Whatever the case, the triggering event initiates the break in tolerance and the cascade of immunological events that eventually lead to the formation of an autoimmune disease begins.Development of autoantibodiesThe third step is the development of autoantibodies and subsequent development of clinical symptoms. Studies have shown that this process can take 3 years or longer and unfortunately, by the time the diagnosis is made, substantial damage to the body may have already occurred.

Currently, the most effective treatment for TTP is therapeutic plasma exchange (TPE). Fresh frozen plasma (FFP), preferably cryoprecipitate-poor plasma (that lacks von Willebrand factor), is used as the replacement fluid in the treatment. The exchange takes place over several days until the patient's platelet count stabilizes above 100 x 109/L.The logic of TPE is to rid the circulation of plasma containing ultra-large vonWillebrand factor (vWF) multimers. vWF is a large multimeric protein that is made by megakaryocytes and endothelial cells. It is is a key factor in platelet adhesion and also is responsible for carrying Factor VIII into the circulation. vWF binds glycoproteins Ib, IIb, and IIIa. The largest multimer is called ultra-large vWF and in normal plasma, it is cleaved into smaller fractions (necessary for balanced coagulation activity) by an enzyme processed by the gene, ADAMTS13. In patients with TTP, the enzyme activity is < 5% of normal and therefore, these ultra-large vWF molecules get into circulation, resulting in excessive platelet aggregation and microvascular thrombus formation.Therapeutic plasma exchange has decreased TTP mortality rate from 90% to 15% since the treatment first came into use as the standard primary treatment of TTP in the 1970's. TPE does not cure TTP, but it arrests the manifestations of the disease until spontaneous remission occurs.

Thalassemias are part of a group of quantitative hemoglobin synthesis disorders in which a defect exists in the rate of production of one or more of the globin chains. This defect results from either a heterozygous or homozygous deletion or inactivation of a globin chain gene.Thalassemias are named according to the affected gene or the globin chain that is showing reduced or absent synthesis.Globin chain loci are found on:chromosome 11 (beta, delta, epsilon, and gamma)chromosome 16 (alpha, and zeta)

Beta thalassemia minor (one gene mutation or deletion). This condition results in a range in beta chain synthesis from 10 - 50%. Beta thalassemia minor exists in several states that are identified with plus or zero as noted earlier in the course. These notations correlate with the degree of gene deletion or inactivation.Because the delta gene is in close proximity to the beta gene, it is included in the beta thalassemia classification.The following pages include illustrations of beta thalassemia states.

Beta thalassemia major, B0/B0 (two gene mutations, deletions or combination) results in very few to no beta chains being produced. Hemoglobin A levels are at or near 0%. (drawing modified from Harmening, 1999) Other genotypes of beta thalassemia major not depicted include B0/B+ (one beta chain deleted, one partially deleted) and B+/B+ (both beta chain genes partially deleted).

In Beta thalassemia minor B+/B one beta gene locus is partially deleted or inactive. With this deletion, only 85% to 95% of the normal level of Hb A is made.(drawing modified from Harmening, 1999)

Occasionally, the beta chain gene deletion extends to include the locus for the delta chain gene. If this deletion occurs on only one chromosome of the pair, it creates delta-beta thalassemia minor. Delta-Beta 0/ BetaHb A and A2 will both be decreased and Hb F will be increased.(drawing modified from Harmening, 1999)

In beta thalassemia intermedia B0/B+, there is one completely deleted or inactive beta chain gene, while the other is partially deleted or inactive. This state also results in Hb A production of 55%-75% of normal.(drawing modified from Harmening, 1999)In beta thalassemia intermedia B0/B, there is one completely deleted or inactive beta chain gene, while the other gene is completely normal. (not shown)

Delta-beta thalassemia major, Delta-beta 0/ Delta-beta0, exists when both gene loci for beta and delta chains are completely deleted or inactive on both chromosomes. In this state, only Hb F can be made (two alpha chains, two gamma chains).(drawing modified from Harmening, 1999)

Retinoblastoma is a tumor arising from retinal tissues of the eye, which can be inherited or spontaneous; the inherited form is associated with a mutation in the Rb1 gene. Retinoblastoma is known to spread into the optic nerve and CSF. This photo shows a retinoblastoma tumor clump. Notice the smaller size of the tumor cells. They are similar in appearance to Acute Lymphocytic Leukemia (ALL), L1 lymphoblasts. Also, it is important to note the clustering of these cells which form a three dimensional aggregate on the cytospin. While a L1 ALL might have similar nuclear:cytoplasmic proportions with similar chromatin and cytoplasmic characteristics, L1 ALL will typically show a liquid distribution pattern on the cytospin with a more even distribution and without clumping.

cTnI is encoded by a separate gene and after translation, a 31-amino acid chain is added to the amino terminal end. This chain makes troponin I cardiac specific. It is measured as an early indicator of an AMI because it is usually released and detectable within 4 - 6 hours following myocardial damage and peaks around 24 hours. It remains elevated in peripheral blood 3 - 7 days and can be elevated as long as 14 days.

As a high percentage of Enterococcus faecium strains carry the Van A gene and are highly resistant to vancomycin. Species identifications are performed in some laboratories where MIC susceptibility testing may not be available. Methods for the phenotypic separation of E. faecium from E. faecalis are limited. Illustrated in this image are positive reactions for acid production from arabinose and melibiose (yellow color), characteristic of E. faecium. E. faecalis are negative for these reactions. A few preformed substrates such as beta galactosidase (E. faecium positive, E. faecalis negative) also serve to separate these two species, accomplished by certain commercial systems that include these substrates. E. faecium is not motile, an additional characteristic helpful to separate vancomycin-resistant Enterococcus species from E. cassiloflavus and E. gallinarum, both of which are motile, and carry the low level resistant gene VAN-c.

A deficiency in one or more coagulation factor will also cause abnormalities in hemostasis. The image to the right depicts the coagulation cascade. Notice how one factor acts upon another to eventually form a stabilized fibrin clot, the end product of the coagulation cascade. Having an abnormally low level, or a complete lack, of a coagulation factor can cause the extrinsic, intrinsic, or common pathways to malfunction, resulting in dangerous hemorrhagic issues including spontaneous bleeding. Two of the most common factor deficiencies are factor VIII (hemophilia A) and factor IX (hemophilia B). Hemophilia A comes in two forms: congenital (inherited) or acquired. Congenital hemophilia A represents the condition where an individual is born defecient (to various degrees) of factor VIII. Acquired hemophilia A is a condition in which an individual spontaneously produced an autoantibody to factor VIII, leaving the body unable to use the factor VIII that may be present. Hemophilia B is an inherited condition where the individual has a mutation of the factor IX gene and is unable to produce adequate levels of this coagulation factor.In some cases, patients have multiple factor deficiencies that are secondary to a primary condition such as vitamin K deficiency, disseminated intravascular coagulation (DIC), and liver disease. With vitamin K deficiency, the liver is unable to produce the coagulation factors that are vitamin K-dependent. During liver disease, the liver may be unable to produce coagulation factors effectively. In DIC, the clotting processes are in overdrive and will consume the coagulation factors that are being produced, leading to low levels of circulating coagulation factors.

Because of the two separation processes, more information and separated solutes can be gained from a sample. The use of two-dimensional electrophoresis is specialized and most applications are in research fields. It is used to study families of proteins in the field of proteomics and protein content in different types of cells. It is also used extensively in genetics to study differences in diseases, gene mutations, and bacterial DNA. In an effort to find ways to detect malignancies earlier, two-dimensional electrophoresis is also used to study tumor cells.

A gene is a hereditary unit or sequence of the nucleotide bases ACGT, occupying a fixed location or locus on the chromosome. It is these genes that carry all the information for life processes.DNA is rewritten into 3 types of RNA, each with a specific task:Messenger RNA (mRNA) carries the protein message to the cytoplasm.Ribosomal RNA (rRNA) is the location of protein synthesis.Transfer RNA (tRNA) is responsible for amino acid transport.Each 3-base nucleotide sequence (codon) codes for a specific amino acid. Some amino acids have more than one codon to direct their placement; this is termed degeneracy.

Each amino acid is coded for by a sequence of three bases. The gene for the beta chain contains the code for a sequence of 146 amino acids. The first six of these amino acids are: valine, histidine, leucine, threonine, proline , and glutamic acid. The specific base sequence for these amino acids is: GTG/CAC/CTG/ACT/CCT/GAG. Normal Human Beta Chain (first six codons) Val HisLeu ThrPro Glu GTGCACCTG ACTCCT GAG Sickle cell hemoglobin (Hemoglobin S) results when, glutamic acid that is normally present in the sixth position on the beta globin chain is substituted with valine.Sickle Cell Hemoglobin (first six codons) ValHisLeu Thr ProVal GTGCACCTGACTCCT GTG "ß6glu-val" in the nomenclature indicates this mutation in the sixth position on the beta chain.

The sickle cell gene is most prevalent in Africa, although it is also common in Mediterranean countries, India, and the Middle East. Less than 2% of African Americans are homozygous for HbS and 8 - 10 % are heterozygous.

Bone marrow transplants may be a cure but currently the risks are too high. Impediments to transplantation include the lack of matched sibling donors and prior transfusions, which have exposed the patient to donor antigens.The best candidates for bone marrow transplants are children less than 16 years old. Two umbilical cord blood transplantations have been performed that reportedly have not remanifested with sickle cell disease.Gene therapy may become an option in the future that may alter the expression of the sickle gene.

The application of DNA analysis to typing blood group antigens started in the early 1990s but is not yet widely available. Molecular methods exist for typing Rh (RHD and RHCE), Kell (K & k), Duffy (Fya & Fyb), and Kidd (Jka &Jkb) loci.In perinatal testing programs, molecular typing can determine the Rh type of the mother, father, and fetus and may be done if the mother has anti-D or another antibody known to cause HDFN. More specifically, if available, DNA methods are typically used in these circumstances: For women who type as weak D in serologic tests, to determine the Rh genotype of the mother to identify if she is partial D or weak D; For women who have made anti-D, to determine the Rh genotype of the father to see if fetal monitoring is needed; For women who have made anti-D, to determine the Rh type of the fetus if the father is heterozygous for RhD or unavailable for testing. Fetal blood typing can be done using fetal DNA from cells obtained by amniocentesis or by testing cell-free, fetal-derived DNA present in maternal plasma at 5 weeks gestation and later. Like all diagnostic methods, DNA typing has limitations and is not 100% sensitive and specific. For example: The blood group's molecular basis may be unknown; Not all alleles in ethnic populations are known; Rare mutations in the RHD and other genes may not be detected; Silencing changes (switching off of a gene) may affect antigen expression; Fetal typing using amniotic fluid may give false-negative results because of maternal cell contamination.

Policies for administering RhIg when the mother's Rh type is weak D vary among countries and within some countries. To understand the issues involved, we need to review the genetics of the Rh D antigen and the types of weak D (formerly Du). The Rh system is complex and only a basic overview will be given.In brief, Rh system inheritance is determined by two sets of genes: RHD codes for the proteins carrying D expression. In most people, the presence or absence of the RHD gene results in being Rh positive or Rh negative, respectively. RHCE codes for different combinations of the proteins carrying CcEe expression and Rh-associated glycoprotein (RhAG). RhAG is needed for the expression of Rh antigens. The RHCE locus is adjacent to RHD on chromosome #1. See a model for the Rh locus (NCBI)D variantsFor a small percentage of people, the inheritance of the RHD gene and the expression of the D antigen may be altered leading to several variants of D encoded by more than 100 RHD alleles

A hemochromatosis gene, HFE, was identified in 1996. Mutations in the HFE gene are found in the majority of patients diagnosed with hereditary hemochromatosis (HH). The locus for the gene is on the long arm of chromosome 6 where it codes for a membrane protein, HFE. The exact mechanism of the role of HFE protein in iron metabolism is incompletely understood. It is thought that HFE, along with a second protein, beta-2 microglobin, interacts with transferrin receptors (TfR) on cell membranes. This interaction supresses the affinity of transferrin for TfR, thus lowering the uptake of transferrin--and its attached iron--into the cell. Transferrin receptors have been found on the surface of a variety of cells, with the greatest concentration on cell membranes of intestinal cells, hepatocytes, and RBC precursors. In addition to HFE, HH is also associated with mutations in other genes involved in iron homeostasis, including hemojuvelin (HJV), TfR, hepcidin, and ferroportin. Hepcidin production is reduced in HH due to all of these genetic causes, with a resulting increase in iron absorption. Mutations in HFE are the most common cause of HH.

Several mutations of the HFE gene have been described. The most common mutation in patients with hereditary hemochromatosis is the C282Y mutation. In the C282Y mutation, a base substitution leads to a change in the amino acid in position 282 from cysteine (C) to tyrosine (Y). The loss of the sulfhydryl-containing amino acid disrupts the tertiary structure of HFE so that it no longer binds to beta-2 microglobulin. Beta-2 microglobulin appears to act along with other proteins to chaperone the newly synthesized HFE out of the Golgi apparatus and to the cell surface where it can then bind to TfR. In the C282Y mutation, HFE remains in the Golgi, never making it to the cell surface. The result is that transferrin binding to TfR is enhanced and excessive amounts of iron enter the cells of the small intestine, liver, and other tissues. A second mutation, H63D, causes a histidine (H) residue in position 63 to be replaced by aspartic acid (D). The mechanism by which this mutation leads to increased iron uptake is less well understood when compared to the C282Y mutation. Unlike the C282Y mutation, the H63D mutation does not seem to affect the binding of beta-2 microglobulin and intracellular movement, since detectable concentrations of the mutated protein are found on cell membranes. Some researchers speculate that the H63D mutation affects the binding of proteins involved in iron regulation and uptake at the cell surface.A third mutation, S65C, leads to a serine-to-cysteine substitution in its associated protein. This mutation has been been found in some compound heterozygotes for C282Y or H63D, but is rarely associated with iron overload in HH.Additional mutations of HFE have been identified, but their clinical significance is unclear. Most laboratories performing molecular assays test for only the C282Y, H63D, and S65C mutations.

The diagnosis of hereditary hemochromatosis (HH) is made through a combination of laboratory tests and medical evaluation of a patient's signs and symptoms. Iron overload is identified by tests that evaluate iron metabolism, while molecular assays are needed to document mutations in the HFE gene or others such as hepcidin, hemojuvelin, or transferrin receptor. Individuals with documented iron overload who exhibit signs and symptoms consistent with HH and who possess HFE or other mutations are considered to have HH. Other causes of secondary iron overload may need to be ruled out.An example of a testing algorithm is shown.

HIV consists of nine genes. Three of the genes provide genetic information for the capsid proteins, envelope proteins, and viral enzymes. The other six genes are regulatory genes, controlling functions such as uncoating of the HIV genome and the penetration of host cells. Gene Number Abbreviation Gene Function 1 gag capsid proteins 2 pol viral enzymes 3 env envelope proteins 4 vif regulatory gene 5 tat regulatory gene 6 vpu regulatory gene 7 nef regulatory gene 8 vpr regulatory gene 9 rev regulatory gene

HIV consists of nine genes. Three of the genes provide genetic information for the capsid proteins, envelope proteins, and viral enzymes. The other six genes are regulatory genes, controlling functions such as uncoating of the HIV genome and the penetration of host cells. Gene NumberAbbreviationGene Function1gagcapsid proteins2polviral enzymes3envenvelope proteins4vifregulatory gene5tatregulatory gene6vpuregulatory gene7nefregulatory gene8vprregulatory gene9revregulatory gene

Tumor suppressor genes and proto-oncogenes regulate cell division. Tumor suppressor genes slow down or stop cell division for repair of DNA damage and promote apoptosis. A mutated tumor suppressor gene results in uncontrolled cell growth, which can be associated with the HPV-related carcinogenisis of cervical cancer. Proto-oncogenes promote cell division, initiate DNA synthesis, and inhibit apoptosis. Mutations occurring in proto-oncogenes convert them to oncogenes which can cause malignancy; this can also play a role in HPV-related malignancies.

Three separate loci (ABO, Hh, and Se) contain the genes that control the location and occurrence of the A and B antigens. Hh and Se genes are closely linked on chromosome 19. The precursor substance is acted upon by the H gene and is converted to H substance. The product of the H gene is an enzyme fucosyltransferase, responsible for attaching fucose to the terminal galactose of the precursor substance on the RBC membrane and thus forming H substance. There are only two recognized alleles at this locus: the active form, H, and an amorph, h. The H gene is a high-incidence gene. People who inherit hh are extremely rare. Since the h gene is amorphic, it does not act on the precursor substance.

As mentioned previously, the A and B genes cannot act directly on the precursor substance. Thus, since individuals with the Bombay phenotype have only the precursor substance and no H antigen, they cannot have A or B antigens, even if they have the A and/or B gene.

The presence of A and/or B antigen on the red cells can be recognized by serological tests with the appropriate antisera so that the presence of the gene that controls its production can be deduced in the absence of both A and B genes (when no A or B antigen is present on the red cells).

The role of leptin in obesity and insulin resistance is sometimes confusing. Some authors refer to leptin as a hormone, not an adipokine. Leptin is synthesized and released from adipose cells in response to adipose tissue changes. It reduces intracellular lipid levels in many types of body cells and thus improves insulin sensitivity. It is an appetite suppressant and inhibitor of fatty liver formation.Leptin is referred to as a "starvation signal" and the leptin gene, is sometimes referred to as "the obesity gene". These names refer to leptin's important function as a messenger in energy metabolism. Leptin signals the hypothalamus when there are increases in fat stores. The hypothalamus then restores metabolic balance by decreasing appetite, stimulating physical activity, and burning of excess calories. During fasting, leptin levels decrease rapidly and hypothalamus signaling results in an increase in cortisol and a decrease in thyroid, sex, and growth hormones. These actions work together to restore energy balance. Leptin is usually increased in obesity, however, similar to increased insulin in obesity, leptin resistance develops. In obesity, appetite suppression does not take place and metabolic rates are lowered. Secreted leptin is not able to stimulate energy balance and healthy caloric intake.

Cepheid was one of the first companies to market an assay for methicillin-resistant Staphylococcus aureus (MRSA), based on its SmartCycler® real-time PCR platform.Molecular detection of methicillin resistance in staphylococci is based on the detection of the mecA gene. However, since coagulase negative staphylococci (CNS) can also possess this gene, discrimination between CNS and MRSA must be achieved by the simultaneous detection of additional gene sequences specific for S. aureus. Cepheid's assay was a multiplex assay that did include targets for six variants of the mecA gene, as well as the S. aureus orfX gene. Despite this, independent investigators documented incidences of both false-positives and false-negatives. The BD GeneOhm™ MRSA assay is another real time assay designed for the SmartCycler® platform. This assay employs molecular beacons for detection. The probe has a hairpin shape, with a fluorophore at one end, and a quencher at the other. In the absence of the target, the hairpin is closed and fluorescence is quenched. In the presence of the target, the hairpin opens when the beacon hybridizes to the target, resulting in the emission of fluorescence, which is measured during each cycle of amplification. Result availability is similar to the Cepheid assay. As with the Cepheid assay, independent investigators documented some incidence of both false positives and false negatives, but noted the advantage of rapid availability of screening results for surveillance purposes.

The 2009 H1N1 influenza virus was first detected in the United States on April 15, 2009.The virus was a unique combination of influenza virus genes never previously identified in either animals or people; they were most closely related to swine-lineage H1N1 viruses (hence the designation of "swine influenza"). However, epidemiological investigations of initial human cases did not identify exposures to pigs and it became apparent that this new virus was circulating among humans and not among U.S. pig herds.By April 21, 2009, the Centers for Disease Control and Prevention (CDC) began working on development of a new vaccine effective against this new strain. On April 24, 2009, the CDC uploaded complete gene sequences of the 2009 H1N1 virus to a publicly accessible international influenza database. At the same time vaccine development was occurring, work was also being done at CDC to help laboratories more quickly identify the 2009 H1N1 virus in patient samples. A real time PCR assay developed by the CDC was cleared for use by the Food and Drug Administration (FDA) under an Emergency Use Authorization (EUA) on April 28, 2009.The development of an effective, rapidly performed molecular assay was critical, because a CDC evaluation of non-molecular rapid influenza assays indicated that while these tests were capable of detecting the novel H1N1 strain when present in high concentrations, the overall sensitivity was low. Positive results with these assays were useful, but negative results did not rule out infection with influenza.

A 2009 evaluation and comparison of a variety of commercially available toxin detection assays, glutamate dehydrogenase (GDH) assays, the cytotoxin assay, cytotoxigenic culture, and real time PCR for the C. difficile tcdB gene revealed that ALL methods demonstrated a relatively low positive predictive value, which compromised the utility of a single test for laboratory diagnosis of C. difficile. However, of all methods, PCR had the highest negative predictive value, and was considered the optimum rapid single test.Molecular methods for C. difficile are based on the detection of the tcd gene. With the application of real time methodology, results can be available within 2 to 3 hours. These methods are highly sensitive and demonstrate good sensitivity, in comparison to all methods with the exception of toxigenic culture. As the methodologies and instrumentation are developed and improved, they are increasingly adaptable to the environment of a busy clinical diagnostic setting. The BD GeneOhm™ and Meridian illumigene® assays are examples of currently available molecular assays for C. difficile.

Meridian's illumigene® assay for C. difficile utilizes loop-mediated isothermal DNA amplification, or LAMP.In standard PCR, primers anneal to single strands of DNA, with polymerase enzyme replicating both strands. In the thermocycling process, denaturing, annealing, and replication occur at different temperatures. The LAMP process is carried out at a constant temperature of approximately 63°C and relies on the strand displacing activity of DNA polymerase. In this assay, a total of six primers are utilized, targeting a conserved region of the tcd gene (the pathogenicity locus, or PaLoc). The activity of the primers and polymerase in the reaction produce strands (copies of the target area) with stem loops at either end. Replicates of the target sequence, of varying lengths, are produced in the amplification process. A byproduct of the amplification is the formation of magnesium pyrophosphate, which forms a white precipitate leading to a turbid reaction solution. The presence of turbidity indicates a positive reaction; the absence of turbidity signifies a negative reaction.The assay requires specific instrumentation, the illumipro-10™, but the foot print is very small. The technical procedure is not difficult, and is amenable for introduction to laboratories with minimal prior experience with molecular methodologies.

Methicillin Resistant Staphylococcus aureus (MRSA) is resistant to the beta-lactam antibiotics. The term methicillin-resistant is historically used to describe resistance to any of this class of antimicrobials even though methicillin is no longer the drug of choice. The acronym MRSA persists and is used interchangeably with ORSA – oxacillin-resistant Staphylococcus aureus. Oxacillin/methicillin resistance implies resistance to all penicillins, cephalosporins, monobactams, carbepenems and beta-lactam/beta-lactamase inhibitor combinations. S. aureus intrinsically produces beta lactamase enzymes that breakdown beta lactam antibiotics (i.e., penicillin); these are designated PBP 1 - 4. The beta-lactam resistance of MRSA is determined by the production of a novel penicillin binding protein called PBP 2' (PBP 2a), that has a reduced binding affinity for beta-lactam antibiotics. This allows MRSA strains to continue cell wall synthesis due to the uninhibited activity of PBP2' even in the presence of otherwise inhibitory concentrations of beta-lactam antibiotics.PBP2' is encoded by a mecA gene located on the MRSA chromosome and is widely distributed among Staphylococcus aureus as well as coagulase-negative staphylococci. The mecA gene is carried by a novel mobile genetic element, designated staphylococcal cassette chromosome mec – SCCmec that is integrated into the bacterial chromosome. The mecA gene is believed to have originated in some coagulase-negative staphylococcal strains and was then transferred into S. aureus, giving rise to MRSA. It is likely that SCCmec serves as the carrier of the mecA gene moving across staphylococcal spp. as these mecA genes have never been found without the presence of a SCCmec-like structure. Phylogenetic analyses of international collections of MRSA and methicillin-susceptible S. aureus isolates have revealed that methicillin resistance has arisen in five distinct lineages designated SCCmec I – V, which differ in both size and genetic composition. In recent years, the gene has continued to evolve so that many MRSA strains are currently resistant to several different antibiotics.

Like methicillin, vancomycin exerts its antimicrobial effect by inhibiting cell wall synthesis, binding irreversibly to cell wall precursors – D-alanyl-D-alanine; and attacking sites responsible for cell wall synthesis. Resistance in VISA strains is thought to be due to: Accelerated peptidoglycan synthesis with increased quantities of D-alanyl-D-alanine residues, which bind & sequester vancomycin molecules Thicker cell walls with reduced peptidoglycan cross-linking (impedes progress of drug molecules) Increased glutamine mucopeptides. All strains with MIC ≥4 µg/ml should be considered candidate VISA strains.Cell wall thickening and transfer of genetic material underlie the development of vancomycin resistance. There is evidence to support the transfer of genetic material among vancomycin-resistant bacterial isolates; the Michigan (2002) VRSA isolate acquired the vanA gene via interspecies transfer from a co-isolated vancomycin-resistant Enterococcus faecalis.

Molecular methods for the detection of tcd region/gene in toxigenic C. difficile are commercially available. Results can be read in as little as 2-3 hours after collection.Molecular methods are highly sensitive and specific; initial studies suggest increased sensitivity over any other available test except culture. These method have relatively rapid turnaround times. Although molecular methods require expensive instrumentation and personnel with the training to correctly perform the techniques, more laboratories are investigating this avenue of diagnostic testing.

In regards to glycopeptide resistance, there are six phenotypes, three of which are more commonly occurring. The VanA phenotype has an inducible high level resistance to vancomycin as well as teicoplanin (encoded by the VanA gene). The VanB phenotype (encoded by two vanB genes) has moderate to high resistance to vancomycin only. The VanC phenotype (encoded by two vanC genes) demonstrates a non-inducible low level resistance to vancomycin. Van A and Van B are the most clinically significant phenotypes and are usually seen among Enterococcus faecalis and E. faecium isolates. Van C is both intrinsic and characteristic in E. gallinarum and E. casseliflavus. Because they are intrinsic rather than acquired, they represent a different impact/significance for hospital epidemiology; definitive speciation can have significance for infection control purposes.At the present time, both ampicillin and vancomycin resistance occur more frequently with E. faecium isolates than with E. faecalis. Most vancomycin resistant E. faecium strains possess the vanA gene.

Oxacillin is the agent of choice for standardized MIC methods (broth & agar dilution). However, since 2006 the Clinical Laboratory Standards Institute (CLSI) has recommended the use of 30 µg cefoxitin disk rather than the oxacillin disk to detect mecA-mediated resistance in the disk diffusion test because the cefoxitin disk test is easier to read and is as sensitive and specific as MIC methods. Results are still reported as "oxacillin-resistant" or "oxacillin-sensitive." Cefoxitin is a better inducer of the mecA gene and gives clearer, easier to read endpoints in disk diffusion tests.The oxacillin disk is read for light growth within the zone of inhibition using transmitted light (plate held up to light), ANY discernible growth within zone of inhibition is indicative of resistance. The cefoxitin disk is read using reflected light.Interpretive Critieria for Cefoxitin Disk Diffusion Test Resisitant Intermediate Susceptible S.aureus/MRSA < 21 mm N/A > 22 mm

To discuss PGx, we must first define two terms - polymorphism and cytochrome P450 (CYP450).A polymorphism is a variation in a gene (allele) that affects at least 1% of the population. CYP450 refers to a family of enzymes found predominantly in the liver. CYP450 enzymes work on a variety of substrates (drugs), altering their chemical structures to facilitate excretion in the urine and feces. There are many known polymorphisms in CYP450 enzymes.

CYP2D6 has received the most attention: It is estimated that about 25% of common drugs are metabolized by CYP2D6. CYP2D6 accounts for only about 1% of all CYP450 enzymes, but it is important in the metabolism of about 100 drugs. There are more than 80 genetic variants that have been described in the CYP2D6 gene. The normal, wild-type allele displays normal metabolic activity whereas some of the variant forms have enhanced or diminished activity. The variants can be grouped generally according to the resulting alterations in protein function. The groupings correlate with four major enzyme metabolic capacities (phenotypes): poor, intermediate, extensive (normal), or ultra-rapid metabolizers.

There are numerous applications for real-time PCR in the laboratory for both diagnostic and research purposes. Diagnostic applicationsReal-time PCR can rapidly detect nucleic acids that are diagnostic of infectious diseases, cancers, and genetic abnormalities. Real-time PCR has allowed for viral quantitation of infectious and newly emerging diseases such as influenza A H1N1 subtype. In malignant diseases, real-time PCR can be performed directly on genomic DNA to detect translocation-specific malignant cells. For RNA samples, real-time PCR has become extremely important for the detection and monitoring of HIV, hepatitis C and CMV. Real-time PCR can also be used for array verification and drug therapy efficacy. Research applicationsIn a research setting, real-time PCR is primarily used to measure gene transcription. The technology is commonly used to determine genetic expression of a particular gene over time in response to different pharmacologic agents or environmental conditions and can also be used to compare gene expression in exposed and unexposed individuals. The use of real-time PCR in this manner can help researchers find and detect diagnostic or prognostic indicators to increase the understanding of disease pathogenesis.

Policies for administering RhIg when the mother's Rh type is weak D vary among countries and within some countries. To understand the issues involved, we need to review the genetics of the Rh D antigen and the types of weak D. The Rh system is complex and only a basic overview will be given.In brief, Rh system inheritance is determined by two sets of genes: RHD codes for the proteins carrying D expression. In most people, the presence or absence of the RHD gene results in being Rh positive or Rh negative, respectively. RHCE codes for different combinations of the proteins carrying CcEe expression and Rh-Associated Glycoprotein (RhAG). RhAG is needed for the expression of Rh antigens. The RHCE locus is adjacent to RHD on chromosome #1. See a model for the Rh locus (NCBI)D variantsFor a small percentage of people, the inheritance of the RHD gene and the expression of the D antigen may be altered leading to several variants of D encoded by more than 100 RHD alleles

The patient's pretransfusion red cells and all donor red cells involved in the case (two group O Rh-negative RBC and four group O Rh-positive red cells initially crossmatched) were phenotyped for Jka.As expected, the patient typed as Jk(a-). The six donor RBC that were incompatible in the initial crossmatch were Jk(a+).The frequency of the Jka gene in Caucasians is ~77%, with most Caucasian red cells (50%) typing as Jk(a+b+).

The influenza A H1N1 2009 virus is regarded as being genetically different from the normally prevelent seasonal influenza A viruses. Novel influenza A virus was originally referred to as "swine flu" because laboratory testing showed that many of the genes in this new virus were very similar to influenza viruses that normally occur in pigs in North America. However, influenza A, subtype H1N1 virus actually has genetic components of human, avian, and swine influenza A combined into one virus; a process called antigenic shift. This virus contains: Two genes from flu viruses from pigs in Europe and Asia One avian, or bird, gene One human geneThis type of virus is called a "quadruple reassortment" virus. Pigs serve as intermediate hosts for influenza A viruses since the respiratory tract of pigs contains receptors for not only swine influenza A viruses, but also avian and human influenza A viruses. Since pigs are also susceptible to avian influenza A viruses from turkeys, ducks, and wild waterfowl, the genetic mixing of swine and avian viruses has been documented over the past 15 years. An additional reassortment may occur with the inclusion of human influenza A genes when pigs are exposed to farmers with respiratory tract infections. The current 2009 H1N1 virus thus represents the most recent example of swine influenza A virus reassortment that contains both avian and human influenza A genes.Reference 1

Chediak-Higashi anomaly is a rare autosomal recessive disorder. It results from a mutation of the gene LYST which encodes a protein with multiple phosphorylation sites. This defect causes a cellular abnormality involving the fusion of cytoplasmic granules. Early in neutrophil maturation normal azurophilic granules form, but they fuse together to form megagranules. Later during the myelocyte stage, normal specific granules form. The mature neutrophils contain both normal specific granules and abnormal azurophilic granules. These large abnormal granules can be seen in the cytoplasm of neutrophils, eosinophils, basophils, monocytes and lymphocytes. These abnormal granules are able to kill bacteria in neutrophils and monocytes; however, the process is much less effective than in normal cells in part, because these neutrophils have impaired locomotion. For these reasons, individuals with Chediak-Higashi have recurrent infections. An accelerated lymphoma-like phase occurs, with lymphadenopathy, hepatosplenomegaly, and pancytopenia. Death often occurs at an early age.