B-Cells and T-Cells

These specialized cells are a critical part of the bodies humoral immune system. They recognize foreign antigens or invaders and mount a quick response. B-cells act quickly by developing antibodies to the antigen epitopes. T-cells react based on what serological class they are in. If it is a CD8 T-cell, its cytotoxic and can quickly fight and phagocytize the antigen, if it is a CD4 T-cell, it works in conjunction with B-cells and other T-cell subclasses to defend the host. This article will dive into B-cells, and every subclass of T-cells and how they work together to form the humoral branch of the immune system.

B-Cells

B-cells also known as B lymphocytes are a type of lymphocyte that functions as part of the humoral component of the adaptive immune system. It’s role is to secrete antibodies, but it also functions as an antigen-presenting cell (APC) that secretes cytokines. It possesses a B-cell receptor (BCR) on its surface that allows it to bind to a specific target antigen and initiate an immune response. B-cells develop from hematopoietic stem cells (HSCs) that originate within the bone marrow. They then develop into multipotent progenitor cells (MPP), which further differentiates into the common lymphoid progenitor (CLP). Development further progresses through several stages through various gene expression patterns and arrangements. Before maturation occurs, positive selection takes place to make sure that the pre-BCR and BCR can recognize and bind to specific ligands through antigen-independent signaling. If the cells are unable to bind, these B-cells cease to develop. Negative selection occurs through binding of self-antigen with the BCR. If the BCR is able to bind self-antigen it undergoes four fates; clonal deletion, receptor editing, anergy, or ignorance. Clonal deletion is the destruction of the B-cell through programmed cell death, in other words known as apoptosis. This is only for those B-cells that have expressed receptors for self-antigens. Receptor editing is exactly what the name suggests; editing of the BCR during the maturation process in an attempt to change the specificity the receptor to not recognize self-antigens. Anergy is used to describe lack of reaction by the bodies immune system. Its a way of saying that the B-cells that express BCRs for self-antigen will simply not be used. The last fate; ignorance means that the B-cell ignores the signal and continues through natural development. When negative selection is complete, the B-cells are now in a state of central tolerance. These mature B-cells do not bind with self antigens. From the bone marrow, B-cells migrate to the spleen as transitional B-cells. Within the spleen they become Follicular B-cells or Marginal zone B-cells depending on the signal received through the BCR. Once completely differentiated, they are now called naive B-cells.

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B-Cell Activation

Activation usually occurs within the secondary lymphoid organs, such as the spleen and the lymph nodes. This is where naive B-cells are positioned once mature. When these naive immunocompetent B-cells encounter an antigen through its BCR, the antigen is internalized by receptor-mediated endocytosis, digested, and positioned on MHC II molecules on the B-cell surface. This allows the B-cell to act as an antigen-presenting cell to T-cells. T-cell dependent activation requires a T-cell helper, most commonly a follicular T-helper cell, to bind to the antigen-complexed MHC II molecule on the B-cell surface through its T-cell receptor (TCR) which drives T-cell activation. These T-cells express the surface protein CD40L and secrete cytokines IL-4, and IL-21 which bind to CD40 on the B-cell surface and act as co-stimulatory factors for B-cell activation. The co-stimulatory factors promote proliferation, immunoglobulin class switching, and somatic hypermutation. Activated T-cells then provide a secondary wave of activation that cause the B-cells to proliferate and form germinal centers. During the production of these germinal centers, activated B-cells may differentiate into plasma blasts, which can produce weak IgM antibodies. Within the germinal centers, B-cells differentiate into high affinity memory B-cells or long-lived plasma cells. The primary function of plasma cells is the secretion of clone-specific antibodies. There are very few antigens that can directly provide T-cell independent B-cell activation. Some components of bacterial cell walls (lipopolysaccharide), and bacterial flagellin are some to name a few. One other mechanism through which B-cell activation is enhanced is through the activity of CD21, CD19, and CD81; all three are surface proteins that form a complex. When the BCR binds to an antigen that is tagged with the complement protein C3, CD21 binds to C3, and downstream signaling lowers the activation threshold of the cell.

Memory B-cell Activation

Activation begins through detection and binding of the target antigen. When the antigen binds, it is taken up by the B-cell through receptor-mediated endocytosis, degraded, and presented onto the MHC II molecule within the B-cell surface. The memory B-cell then acts as an antigen-presenting cell that presents the antigen:MHC II complex to T-cells. Most commonly memory follicular T-helper cells that bind through their TCR. The memory B-cell is then activated and differentiates into either plasmablasts and plasma cells or generate germinal centers.

T-Cells

A T-cell is another lymphocyte, which is a subset of white blood cells. They are called T-cells because they mature in the thymus from thymocytes. There are several subsets of T-cells, each with a specific role in the immune system. These T-cells, just like B-cells originate from hematopoietic stem cells in the bone marrow. These lymphoid progenitor cells populate the thymus and expand by cell division to immature thymocytes. The earliest thymocytes do not express either CD4+ or CD8+ and are classified as double negative cells. Through progression they become double positive and then eventually differentiate into single positive cells, either becoming CD8+, or CD4+. Its interesting to note that there is a small population of double positive T-cells within the peripheral circulation, although their function is unknown. About 98% of thymocytes undergo apoptosis during the development process by failing either positive selection or negative selection. The 2% that survive leave the thymus and become mature immunocompetent T-cells. Lets review positive and negative selection again. Positive selection selects for T-cells that are capable of interacting with MHC molecules. During positive selection signals by double positive precursors express either MHC class I or II receptors. A thymocytes fate is determined during positive selection. Double positive CD4+/CD8+ cells that interact with MHC class II molecules eventually become CD4+ cells, and on the contrary thymocytes that interact well with MHC class I molecules mature into CD8+ cells. Negative selection removes thymocytes that are capable of strongly binding with self MHC peptides.

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T-Helper Cells

T-helper cells do just what their name suggests, they help other cells in immunological processes. This is evident in the activation of B-cells talked about previously. These cells are also most well known as CD4+ T-cells because the highly express CD4 glycoprotein on their surfaces. These T-cells become activated when they are presented with peptide antigens or epitopes by MHC class II molecules, usually present on antigen-presenting cells. Once activated, these cells proliferate rapidly and secrete multiple cytokines. T-helper cells differentiate into several subtypes; TH1, TH2, TH3, TH17, TH9, and THF, each secreting different cytokines to facilitate different pathways of the immune response. This is an article for another time.

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Cytotoxic T-Cells

These killer T-cells destroy virus-infected cells and tumor cells. These cells are known as CD8+ T-cells since they express the CD8 glycoprotein on their surface. These cells recognize targets by binding to antigen epitopes that are associated with MHC class I molecules. Cytotoxic T-cells are highly regulated by Regulatory T-cells through IL-10, adenosine, and other molecules. They can be inactivated to an anergic state, which prevents autoimmune diseases.

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Memory T-Cells

These memory T-cells are long-lived and when presented with an antigen that is recognized they can quickly expand and differentiate into large numbers of effector T-cells. These memory T-cells can either be CD4+ or CD8+ T-cells. There are four subtypes of memory T-cells that will be discussed below.

Central memory T-cells express CD45RO, C-C chemokine receptor type 7 (CCR7) and L-selectin which are all surface protein markers. They have high expression of CD44, and is commonly found within the lymph nodes.

Effector memory T-cells express CD45RO, but lack expression of CCR7 and L-selectin. These T-cells also have high expression of CD44, but are not found in the lymph nodes. These T-cells are found in the peripheral circulation and tissues.

Tissue resident memory T-cells occupy tissues without recirculating. The one specific surface marker that is associated with these cells is integral aeB7.

Virtual memory T-cells differ from all other memory subsets in that they do not originate from a clonal expansion event. These cells reside at low frequencies.

Natural Killer T-cells (NK)

First off, it should be mentioned that these cells should not be confused with natural killer cells of the innate immune system. Unlike conventional T-cells that recognize antigen epitopes presented on MHC I/II molecules, NKT cells recognize glycolipid antigens presented by a molecule called CD1d. When these cells are activated, these cells perform functions from both T-helper cells and cytotoxic T-cells. These cells specialize in recognizing tumor cells and cells infected with herpes viruses.

 

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Use of Selective and Differential Media

There are many different types of media that are used in a microbiology laboratory. Generally speaking there are three types of media used; selective, differential, and supportive or nutritive.

Selective media are manufactured to support the growth of one type of microorganism while inhibiting the growth of another, in other words, it selectively grows one type of microorganism. It is not uncommon or this media to contain antimicrobials, dyes such as crystal violet, or even alcohol. Some of the more routine selective media types are EMB agar, mannitol salt agar, MAC, and a PEA agar.

Differential media is used to distinguish microorganisms from one another based on growth characteristics that are evident when growth is obtained. There are visible differences between microorganisms when growth is achieved. One fo the more common differential media used is the MacConkey agar. This differentiates between lactose fermenters and non-lactose fermenters.

Supportive media is used to support the growth of a wide range of microorganisms. They are typically non-selective because they want to achieve growth of a wide array of microorganisms. Some more common ones include the sheeps blood agar, as well as the chocolate agar. It has the added X and V factors to support the growth of Haemophilus influenzae.

Cells Cells Cells!

The immune system is the host defense system against foreign pathogens. It is an extremely adept system comprised of the innate immune system and the adaptive immune system, as well as complement. For more information on those two systems as a whole, review part one of the immune system.

This part of the immune system overview will focus on the leukocytes or granulocytes of the innate immune system.

The most abundant leukocyte is the neutrophil. It comprises about 40-70% of the white cells an individual has. The maturation of the neutrophil is myeloblast, promyelocyte, myelocyte, metamyelocyte, band neutrophil, segmented neutrophil. The cytokine responsible for stimulating neutrophil production in the bone marrow is G-CSF (granulocyte colony stimulating factor). There are three pools in the bone marrow, the stem cell pool, consisting of HSCs, the proliferative pool, full of mitotic cells, and the maturation (storage) pool. Full of metamyelocytes, bands, and PMNs. During the proliferative pool stage GM-CSF, G-CSF and IL-3 are all used as growth factors to help the neutrophil differentiate and mature. Granulocyte release from the bone marrow is stimulated by G-CSF. Once in circulation neutrophils are divided randomly into either a circulating pool and a marginated pool. The neutrophils in the marginated pool are loosely localized to the walls of capillaries in tissues. Neutrophils can move freely between the two pools. Integrins and selectins are important as they allow neutrophils to marginate and allow them to move into the tissues by using diapedesis. Diapedesis is the extravasation of blood cells through intact vessel walls.

In response to inflammatory mediators and chemoattractants the neutrophil is activated and it results in reorganization of the actin cytoskeleton, membrane ruffling, adhesion and motility. In basic terms, when an infection is ongoing, the surrounding cells and tissue release cytokines and inflammatory mediators that attract neutrophils specifically to come to the site and help control the infection. Chemotaxis is the term for this. The neutrophil attaches to the substratum (endothelial surface) which allows extensions of pseudopods to attach through integrins. Contraction allows the cell body to be pulled forward (still attached). Release of the neutrophil at the back allows the cell to move forward.

Once the neutrophil has reached the site of infection it aids in fighting the infection or pathogen by phagocytosis. Phagocytosis occurs when a neutrophil surface receptor recognizes an antigen either through direct recognition, or to recognize an opsonized antigen. An opsonized antigen remember is when particular cellular processes, such as complement, present pathogenic antigens to these neutrophils to aid in phagocytosis so the neutrophil doesn’t have to search for the pathogen. With recognition comes attachment and engulfment. Cytoplasmic pseudopodia surround the particle forming a phagosome within the neutrophil cytoplasm. The formation of the phagosome allows the NADPH oxidase complex to form which leads to the generation of reactive oxygen species (ROS) such as hydrogen peroxide which is converted to hypochlorite by myeloperoxidase. (O2 dependent). A series of metabolic changes can occur like the changing of the pH and that allows primary or secondary granules within the neutrophil to release numerous bactericidal molecules into the phagosome. (O2-Independent). Bactericidal molecules aid in the killing of foreign pathogens. There is a third mechanism to which neutrophils are able to fight off foreign invader and its by using NETS. Neutrophils can generate an extracellular net that consists of chains of nucleosomes from unfolded nuclear chromatin. These structures have enzymes from neutrophil granules and can trap and kill some gram positive and gram negative bacteria, and fungi. NETs are generated at the time neutrophils die.

Monocyte development is similar to that of neutrophilic maturation because they are both derived from the granulocyte monocyte progenitor. M-CSF (macrophage colony stimulating factor) is the major cytokine responsible for the growth and differentiation of monocytes. Once in the tissue, monocytes differentiate into macrophages, depending on the tissue that the monocytes migrate too, for example, in the lymph nodes they differentiate into dendritic cells, and in the liver, they differentiate into Kupffer cells.

Eosinophils make up 1-3% of the cells in the bone marrow. Eosinophil granules are full of synthesized proteins, cytokines, chemokines, growth factors and cationic proteins. Degranulation can occur in multiple ways; by classic exocytosis, granules move to and fuse with the plasma membrane and the granules secrete into the ECS (Extracellular Space). By compound exocytosis, the granules fuse in the cytoplasm before moving to the plasma membrane. Piecemeal degranulation is when vesicles remove specific proteins from the secondary granules and then migrate to the plasma membrane and then emptying into the ECS. Eosinophils play a role in immune regulation. Eosinophils secrete major basic protein (MBP) which is the cause of mast cell degranulation and cytokine production. Eosinophils are implicated in both type 1 and type 2 immune response, primarily being infectious diseases. Eosinophils are primarily implicated in parasitic infections and are the hallmark characteristic of helminth infections. They help drive antibody production and suppress phagocytosis by secreting arylsufatase which inactivated leukotrienes and secrete antihistamine which counteracts the action of mast cells and basophils.

Basophils and Mast cells are usually grouped together, although basophils are a true WBC because they mature in the bone marrow and circulate in the blood with granules. Mast cell precursors leave the bone marrow and migrate to a tissue where they mature. Basophils and mast cells have membrane bound IgE on their surface. When activated by an antigen causes degranulation (histamine and heparin, which leads to an inflammation causing vasodilation and edema. They also secrete cytokines that activate B and T cells. Basophils are capable of releasing large quantities of subtype 2 helper T cell cytokines such as IL-4, and IL-13 that regulate the TH2 immune response. Mast cells function in chronic allergic reactions, Basophils are the initiators of allergic inflammation through the release of preformed cytokines. Basophils can play a rule in angiogenesis through the release of VEGF and its receptors.

To recap everything that has been learned with this article; neutrophils are the most abundant leukocyte encountered and play a huge role in the innate immune system. They are often the first to a site of infection or inflammation and use multiple mechanics of phagocytosis to control the situation. Monocytes circulate and settle into a tissue where they become resident macrophages. Macrophages also phagocytize foreign antigens when it comes into contact with the tissue they reside in. Eosinophils are active in infections, particularly of parasitic origin. Eosinophilia is a common finding in helminth infections. Basophils and mast cells work in conjunction with IgE to mediate hypersensitivity allergic reactions. They are the ones to thank for making your nose run.

Overview of the Immune System; Part One

The overall function of the immune system is to prevent or limit infection. It is essential for survival. Multiple organ systems, cells, and proteins are involved in the immune response. It is the most complex system that the human body has. The immune system is differentiated into two directions. Innate or non-specific immunity or Acquired (specific) immunity.

The Innate immune system consists of many components. The skin acts as a mechanical barrier and is typically the first line of defense against foreign substances. Mucous membranes consist of the bodies normal microbiota which compete with invading microbes. The mucous membranes are also lined with mucous and cilia which act in an elevator type motion to push foreign substances away. Physiological barriers such as temperature, pH and the complement system. The more acidic environment that a lower pH offers disrupts bacterial growth. Antimicrobial proteins and peptides are present in different epithelial locations in the body. Lysozymes are present in the tears and saliva and cleave the peptidoglycan cell wall present in bacteria. Secretory phospholipase A2 is present in the gut and can enter the bacterial cell and hydrolyze lipids in the cell membrane. Lectins target gram positive bacteria and forms pores in the membranes. Defensins integrate into the lipid and form pores which causes loss of membrane integrity. These defensins are present in PMNs (neutrophils) and lamellar bodies in the gut. Cathelicidins are present in neutrophils and macrophages in the lungs and intestines and distrupt membranes. Histatins are constitutively produced by the glands in the oral cavity and are active against pathogenic fungi.  Inflammation plays a huge role in the Innate immune system. Inflammation induces vasodilation and increase in capillary permeability causing an influx of immune cells like PMNs and macrophages. Inflammation can be observed by the four cardinal signs; rumor (redness), tumor (swelling), color (heat), and dolor (pain). The innate immune response is a rapid response.

Innate Immunity

The complement system recognizes features of microbial surfaces and marks them for destruction by coating them with C3b. There are three distinct pathways; the classical pathway, the lectin pathway, and the alternative pathway. All pathways generate a C3 convertase which cleaves C3, leaving C3b bound to the microbial surface and releasing C3a. In the classical pathway the activated C1s cleaves C4 to C4a and C4b which binds to the microbial surface. C4b then binds C2, which is cleaved by C1s to C2a and C2b forming the C4b2b complex. C4b2b on the microbial surface is an active C3 convertase which cleaves C3 to C3a and C3b. This results in opsonization of the bacterial surface by C3b. The C4b2b3b complex is an active C5 convertase leading to the development of the membrane-attack complex. Each complement component (C4a/b, C2a/b, C3a/b) have different functions, but that is another discussion for another time. The lectin pathway of complement activation is when mannose-binding lectin (MBL) and ficolins recognize and bind to carbohydrates on the pathogen surface. Ficolins are similar to MBLs, but have a different carbohydrate binding domain. MBLs bind with high affinity to mannose and fucose residues. Conversely ficolins bind oligosaccharides containing acetylated sugars. When MBL binds to a pathogen surface MBL-associated serine protease (MASP)-2 is activated and cleaves C4 and C2 similar to the classical pathway. The alternative pathway is an amplification loop for C3b formation that is accelerated by properdin (factor P) in the presence of pathogens. Properdin stabilizes the C3bBb complex. C3 undergoes spontaneous hydrolysis to C3(H20) which binds to factor B, allowing it to be cleaved by factor D into Ba and Bb. The C3(H20)Bb complex is essentially a C3 convertase which cleaves more C3 into C3a and C3b. C3b molecules result in opsonization of bacterial surfaces. Its important to recognize that all pathways lead to generation of a C5 convertase. C4b2a4b in the classical pathway, C4b2a3b in the lectin pathway, and C3b2Bb in the alternative pathway. C5 is cleaved into C5a/b that initiates the assembly of the terminal complement components. These are the terminal complement components that form the membrane-attack complex.

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The membrane attack complex consists of an assembly of C6, C7, and C8. This complex undergoes a conformational change that results in polymerization of C9 which generates a large pore in the cell membrane. Host cells contain CD59 which prevents the assembly of the C9 molecules preventing the formation of the membrane-attack complex.

C3a, C4a, and C5a are unique in that these complement components are called anaphylatoxics. They initiate a local inflammatory response when systemic injection of these molecules occurs. They induce smooth muscle cell contraction and increased vascular permeability. They induce adhesion molecules and activate mast cells that invade and populate submucosal tissues to release inflammatory mediators such as histamine and TNF-a.

The Acquired or adaptive immune system is all about specificity. The Humoral branch of the acquired immune system is executed by the B lymphocytes that produce antibodies to specific antigens. The cell-mediated branch consists of antigen presenting cells (APC) such as the dendritic cells processing foreign substances and presenting proteins of those substances as antigens through the major histocompatibility complex (MHC) to CD8 T lymphocytes. These are cytotoxic T-cells that kill these foreign antigens. The acquired immune response is a slow response because it takes the body time to produce antibodies. An important aspect of the adaptive response is memory. Once antibodies have been produced to an antigen, these responses last and the time it takes to produce an antibody on subsequent exposures is rapidly decreased.

These two different systems work in conjunction to produce an adequate and sustained response. When foreign antigens are processed and expressed on the surface of APCs as MHC peptides, pro-inflammatory cytokines such as IL-12p70, IL-18, and IFN-a are secreted. These attract NK cells which primarily attack viruses as well as PMNs and macrophages that phagocytize these antigen peptides to destroy them. Adaptive immunity is also started with dendritic cells that also undergo antigen uptake and processing. This is also called the maturation signal. This signal is augmented by IFN-y and TNF-a secreted by macrophages and NK cells. These dendritic cells either present the antigen to B lymphocytes which are the antibody producers or they present the antigen to CD4/CD8 T-cell lymphocytes.

There are multiple classes of antibodies. IgD is typically expressed on B-cell lymphocytes during differentiation with IgM. IgD is also present in the serum in low concentrations. IgM is a pentamer and the largest immunoglobulin. It is the first antibody that is produced in the immune response. IgA is in high concentration in the mucosal linings, saliva, and tears. Typically part of first line defenses. IgG is present in high concentrations in the serum. IgG is unique in that it can cross the placenta. IgE is involved in allergic reactions. It binds to mast cells and basophils causing degranulation.

-Caleb

Streptococcus Identification and Work Up

Streptococcus is a genus of coccus gram positive bacteria. They typically grow in chains of pairs as cell division occurs along a single axis in this family of bacteria. Most species in the streptococcus genus are oxidase negative, catalase negative, and most are facultative anaerobes. Many streptococcal species are not intact pathogenic and are part of the commensal human microbiota of the skin, mouth, intestine and upper respiratory tract. However, certain streptococcus species are the pathogenic agent for many cases of pink eye, meningitis, bacterial pneumonia, endocarditis, erysipelas, and necrotizing fasciitis.

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Species of Streptococcus are classified based on their hemolytic properties. Alpha-hemolytic species cause oxidation of the iron within the red cells, giving a greenish color to appear on the blood agar. The bacteria produces hydrogen peroxide which oxidizes the hemoglobin to biliverdin. This is also referred to as incomplete hemolysis or partial hemolysis. Beta-hemolytic streptococci cause complete degradation of the red cells, appearing as wide clear areas surrounding the bacterial colonies. Gamma-hemolytic species cause no hemolysis.

The beta-hemolytic species are further classified by the Lancefield grouping. This is a serotype classification that describes the specific carbohydrate present on the bacterial cell wall. There are serotypes for Lancefield groups A-V. For times sake, the only ones that will be discussed are Group A, and Group B.

Alpha-Hemolytic Strep

Strep pneumoniae, often referred to as pneumococcus is the leading cause of bacterial pneumonia. It can also be the etiological agent for otitis media, sinusitis, meningitis and peritonitis. The viridian’s group of alpha-hemolytic streptococci are a large group of commensal organisms. They possess no Lancefield antigens (carbohydrates) and can or can not be hemolytic.

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Beta-Hemolytic Strep

Streptolysin which is an exotoxin is the enzyme produced by the strep species that causes complete hemolysis of the red cells in the media. There are two types of streptolysin; Streptolysin O (SLO) and Streptolysin S (SLS). Streptolysin O is an oxygen-sensitive (labile) cytotoxin which is secreted by most of the Group A Streptococcus (GAS) which interacts with cholesterol in the membrane of the red cells. Streptolysin S is an oxygen-stable cytotoxin also produced by GAS species that affects the innate immune system of the host. It works in preventing the host immune system from clearing the infection.

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Group A Strep

Group A strep, otherwise known as S. pyogenes is the causative agent for strep infections, invasive and non-invasive. The most common infection is pharyngitis, otherwise known as strep throat, impetigo, and scarlet fever. All these infections are non-invasive. Invasive GAS infections include necrotizing fasciitis, pneumonia, and bacteremia (bacteria present in the blood). Complications can arise from GAS infections. Rheumatic fever is a disease that affects the joints, kidneys, and the heart valves. It is the consequence of an untreated strep A infection. Antibodies created by the immune system cross-react with proteins in the body which causes an immune-mediated attack on the hosts own cells. Essentially an acquired auto-immune disease. GAS is also implicated in pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections (PANDAS). Autoimmune antibodies affect the basal ganglia causing rapid onset of psychiatric, motor, sleep and other neurological symptoms, primarily in the pediatric population.

GAS is diagnosed by either an rapid strep test or by culture.

Group B Strep

GBS, otherwise known as S. agalactiae, causes pneumonia and more importantly meningitis in neonates and the elderly. The American Congress of Obstetricians and Gynecologists now recommends that all women between 35 and 37 weeks gestation to be tested for group B strep. Those who test positive should be given prophylactic antibiotics during labor. The bacteria can cause premature rupture of the membranes during pregnancy and can colonize and cross the placenta to infect the fetus.

Laboratory diagnosis and Workup

After initial culture and preliminary identification of beta-hemolytic streptococcus is suspected there are further identification tests to make an accurate diagnosis. The first is by Lancefield antigen determination. Commercially available Lancefield antisera is used for the differentiation of beta-hemolytic species. These usually come as small kits and are directed to Lancefield groups A, B, C, F, and G. Antigen detection is demonstrated by agglutination by specific antibodies that are provided.

The PYR test is a rapid colormetric method most often used to distinguish S. pyogenes from other beta-hemolytic species. The PYR tests for the presence of the enzyme pyrrolidonyl aminopeptidase. This enzyme hydrolyzes L-pyrrolidonyl-b-naphthylamide (PYR) to B-naphthylamide, which produces a red color when a specific reagent is added. The test can be performed on paper strips that contain dried chromogenic substrates for the pyrrolidonyl aminopeptidase. S. pyogenes is PYR positive and in the case of an unknown organism displaying S. pyogenes morphology plus being PYR positive, it is acceptable to presumptively identify as S. pyogenes.

Bacitracin susceptibility is another test that can be used to differentiate S. pyogenes from other beta-hemolytic strep species. S. pyogenes has an increased susceptibility to bacitracin. A pure culture must be obtained and streaked on a sheep blood agar plate. A small disk containing 0.04 U of bacitracin is placed on the plate and incubated overnight at 35 degrees celsius in 5% CO2. A zone of inhibition surrounding the disk indicates susceptibility.

With the introduction and advancement of nucleic acid detection and serological methods in the laboratory it is now getting easier and faster to detect strep species without relying on culturing and further confirmation testing. It should be important to note that this does not replace the use of a culture or any of the further testing mentioned above. Serological testing relies on antibodies to anti-streptolysin O and anti-DNase B. The antibody levels against streptolysin O rise within one week of infection and peak around 3-6 weeks. DNase B is a nuclease among many that S. pyogenes uses to escape neutrophil extracellular nets. DNase B is specific for S. pyogenes. The antibody levels to DNase B rise post two weeks infection and reach maximum titers at around 6-8 weeks.

The Optochin test is used to differentiate alpha-hemolytic streptococci. This is either S. pneumoniae or strep viridians. S. pneumoniae species are sensitive to the chemical ethylhydrocupreine hydrochloride, otherwise known as optochin. Optochin disks, sometimes called P disks can be obtained from a commercial vendor. A pure culture is obtain and incubated to allow growth overnight. When growth has occurred, a subculture is plated and a P disk is placed on the agar and incubated overnight at 35 degrees celsius with 5% CO2. The zone of inhibition is then measured and a zone greater than 14 mm indicates sensitivity and allows for the presumptive diagnosis of pneumococci.

The bile solubility test is usually used as confirmatory test for S. pneumoniae that distinguishes it from other alpha-hemolytic species. Sodium deoxycholate (Bile) will lyse the cell wall of pneumococcal species.

-Caleb