KINDS OF ZYGOMYCETES:/ SporangiophoreRhizopus: Although this genera can cause disease, they are also commonly found as a contaminant. Colonies grow rapidly and resemble cotton candy. Colonies darken with age, becoming gray or yellow-

brown. The reverse is white. Mycelia are marked by numerous stolons connecting groups of long sporangiophores. Sporangiophores are usually unbranched, long, and terminate in a columella and a dark round sporangium

containing oval colorless to brown spores. Stolons bear large rhizoids which are found immediately adjacent to the sporangiophore in the nodal

position. Columella and sporangium collapse easily after discharging spores.

Absidia: Colonies mature rapidly and resemble coarse, gray wool or cotton candy. The reverse is white or light gray. Frequently considered a contaminant in cultures, Absidia species are similar in microscopic appearance to

Rhizopus, but rhizoids are internodal. Rhizoids may be difficult to find, as in the case illustrated below. Sporangia are slightly elongated spheres ranging from 20-120μ in diameter. Sporangiospores are round to oval

and measure 3-4.5μ.

Cunninghamella: Frequently considered a contaminant in cultures, Cunninghamella species have broad, almost aseptate

hyphae and long, branched sporangiophores that end in swollen vesicles that are covered with spine-like denticles.

Attached to the denticles are round-oval sporangiolum that range in size from 6-14μ.

Mucor: A common contaminant, but may cause invasive disease. Colonies are fast-growing and resemble white-to-gray cotton candy, darkening with time. The reverse is light-colored to white. Hyphae are wide, like other Zygomycetes, usually approximately 6-15μ. No rhizoids are formed. Sporangiophores are long, often branch, and bear large (50-300μ), round sporangia.

DsDNA Crithidia luciliae test is an indirect immunofluorescent antibody test employing the hemoflagellate, Crithidia luciliae, as a substrate. This single-cell organism possesses a giant mitochondrion containing a highly condensed mass of circular dsDNA.1 This mass of dsDNA, known as the kinetoplast, appears to be free of histones or other mammalian nuclear antigens.1,2 It serves as a sensitive and specific substrate for detecting autoantibodies to dsDNA.Autoantibodies to dsDNA occur almost exclusively in patients with systemic lupus erythematosus (SLE) and as such are considered marker antibodies.

LYSINE IRON AGAR.o Lysine DEAMINATION – aerobic-slant.

(+) dark red color (-) purpleo Lysine DECARBOXYLATION- anaerobic- butt

(+) purple color (-) yellow

Types of Conidiophore Formation

1. Geotrichum candidum Some species form conidia on single, unbranched hyphae. Colonies are fast growing, flat, white to cream, dry and finely suede-like with no reverse pigment. Hyphae are hyaline, septate, branched and break up

into chains of hyaline, smooth, one-celled, subglobose to cylindrical arthroconidia. Arthroconidia are 6-12 x 3-6 µm in size and are released by the separation of a double septum. Used in cheese making

2. Trichoderma viride is a fungus and a biofungicide. It is also a pathogen in its own right, causing green mould rot of onion. Example of a branching pattern of conidiophores bearing clusters of conidia at their

tips.

3. Penicillium claviforme Species of Penicillium are recognized by their dense brush-like spore-bearing structures called penicilli (sing.: penicillus). The

conidiophores are simple or branched and are terminated by clusters of flask-shaped phialides. The spores (conidia) are produced in dry chains from the tips of the phialides, with the youngest spore at the base of the chain, and are nearly always green. Brush like

4. Phoma species Colonies are spreading, greyish-brown, powdery or suede-like and produce large,

globose, membranous to leathery, darkly pigmented, ostiolate pycnidia. Conidia are produced in abundance within the pycnidia on narrow thread-like phialides, which are hardly differentiated from the inner pycnidial wall cells. A flask-shaped structure with conidiophores developing from cells of the pycnidial wall.

Phaeohyphomycosis is a cosmopolitan disease. Patients are usually adults, and about half of them seem to be immunologically compromised by associated underlying diseases such as diabetes mellitus, tuberculosis, leprosy, acquired immunodeficiency syndrome (AIDS), lymphoma, and leukemia. Causative agents are: a. Exophiala jeanselmei, Exophiala moniliae and Wangiella (Exophiala) dermatitidisb. Alternaria alternate – paranasal sinusitis, skin, soft tissue, cornea, lower respiratory tract, peritoneumc. Curvularia lunata – Endocarditis, catheter site infections, nasal septum, paranasal sinuses, cornead. Cladophialophora bantiana/ Cladosporium- deep infections, 44% of cerebral PHM infections are caused by.

Dematiaceous FungiThe term "dematiaceous" refers to the characteristic dark appearance of this group of fungi as it grows on agar. Colonies are dark gray, brown, or black and, importantly, have a black reverse when the bottom of the agar plate is examined. This distinguishes the dematiaceous fungi from fungi with black conidia but an otherwise pale mycelium, such as A. niger.Three groups based on morphology in slide culture:

1. Dematiaceous Fungi with Muriform Macroconidia - A conidium with both longitudinal and transverse septa Alternaria

Colonies mature rapidly, are wooly or cottony, and have a black reverse and a gray-white surface that becomes green-black or brown with a light border as the colony agesConidia form chains or occur singly and are divided by transverse and longitudinal septations; conidia usually have a club-shaped configuration Identification beyond the genus level is difficult

Ulocladium Usually considered a contaminant Colonies mature rapidly and are typically wooly or cottony and dark brown to black; the reverse is black Conidia more spherical than Alternaria, do not form chains, and are borne on short, geniculate

conidiophores

Epicoccum Usually considered a contaminant Colonies grow fairly rapidly and progress from cottony yellow or orange to brown or black. The reverse

may be red and diffusible yellow, red, brown, or orange pigment may be present Short conidiophores arise from sporodochia - A cushion-shaped mass of hyphae bearing

conidiophores. Muriform macroconidia are spherical to club shaped

2. Dematiaceous Fungi with Transversely Septated(with compartments) Macroconidia Curvularia

May cause fungal sinusitis and keratitis, endocarditis, pulmonary infections, and subcutaneous phaeohyphomycosis; may also be contaminants

Colonies dark olive green to brown or black; surface pink-gray and wooly; the reverse is dark Macroconidia characteristically 4-5 cells separated by transverse septae which arise sympodially from twisted conidiophores;

macroconidia have a characteristic curved appearance secondary to disproportional enlargement of a central cell Drechslera

Not reported as a pathogen Produce cylindrical, multicelled macroconidia from geniculate conidiophores Resemble Bipolaris species; may be distinguished in saline mounts by germ tubes extending from the

conidium between base and septum at right angle to the long axis of the macroconidium. In contrast, Bipolaris germ tubes arise from both ends of the base of the conidium and extend parallel to the long axis of the macroconidium

3. Dematiaceous Fungi with Microconidia Cladosporium

May cause cutaneous, subcutaneous, and eye infections, but are among the most common dematiaceous mold contaminants

Nomenclature is changing, as with the rest of medical microbiology: Cladosporium carrionii may currently be better named Cladophialophora carrionii , but Koneman et al. elect to use the prior term in their popular textbook

Colonies are dark with a black reverse and may show velvety gray, gray-green, or purple-brown grown on a flat, dark base. Elliptic microconidia are found in long chains.

Cladosporium-type conidiation displays freely branching hyphae that bear long chains of dark-staining, elliptical conidia that often contains scars or dysjunctors at the sites of attachment

Phialophora verrucosa Causes chromoblastomycosis, phaeohyphomycosis, or mycetoma Colonies are slow growing, dark gray or black, and hairlike Characteristic urn-shaped phialides bear tight clusters of spherical to elliptical, hyaline conidia

The genus Syncephalastrum is characterised by the formation of cylindrical merosporangia on a terminal swelling of the sporangiophore. It can cause nail disease, especially in damaged nails.

Target Vs Signal AmplificationNucleic acids can be detected using either target or signal amplification methods. Briefly, target amplification enzymati-cally increases the number of target molecules. In short, "at the end of the day," there are more molecules of the targeted nucleic acid. In contrast, signal amplification does not increase the target but uses highly sensitive reporter molecules or probes to detect the target. For example, in signal amplification, the same number of molecules exists at the end of the day, but the molecular methods act as a kind of "magnifying glass" to aid in their "visualization."

a. TARGET AMPLIFICATION Target amplification is the most frequently used method and is accomplished using several technologies. PCR, transcription-mediated amplification (TMA), nucleic acid sequence based amplification (NASBA), rolling cycle amplification (RCA), ligase chain reaction (LCR), and strand displacement amplification (SDA). All of these methods use a polymerase or ligase and short synthetic oligonucleotides known as primers. The primers specifically bind to complementary sequences found in the infectious agent.

b. ISOTHERMAL AMPLIFICATION Isothermal methods acquired their name after the Latin meaning of "iso" being same or similar and after the Greek "thermal (therme)" meaning heat. Isothermal amplification can be further subdivided into methods that target single-dimensional linear nucleic acid targets vs methods that require two-dimensional targets or probes (i.e., circular molecules). Isothermal approaches do not require thermocyclers and is one of the advantages of these methods. In addition, these methods have a high throughput because all steps can be performed in a single tube.

branched DNA assay is a signal amplification assay (as opposed to a target amplification assay) that is used to detect nucleic acid molecules. At this point, signal amplification takes place. A label extender DNA molecule is added that has two domains (similar to the first extender). The label extender hybridizes to the target and to a pre-amplified molecule. The preamplifier molecule has two domains. First, it binds to the label extender and second, it binds to the amplifier molecule. An example amplifier molecule is an oligonucleotide chain bound to the enzyme alkaline phosphatase.

The thermal cycler (also known as a thermocycler, PCR machine or DNA amplifier) is a laboratory apparatus most commonly used to amplify segments of DNA via the polymerase chain reaction (PCR). Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase (an enzyme originally isolated from the bacterium Thermus aquaticus). This DNA polymerase enzymatically assembles a new DNA strand from DNA building-blocks, the nucleotides, by using single-stranded DNA as a template and DNA oligonucleotides (also called DNA primers), which are required for initiation of DNA synthesis. The vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample through a defined series of temperature steps.

Immunofluorescence Immunofluorescence is a technique allowing the visualization of a specific protein or antigen in tissue sections by binding a specific antibody

chemically conjugated with a fluorescent dye such as fluorescein isothiocyanate (FITC). The specific antibodies are labeled with a compound (FITC) that makes them glow an apple-green color when observed microscopically under

ultraviolet light. There are two major types of immunofluorescence staining methods: •

o 1) direct immunofluorescence: staining in which the primary antibody is labeled with fluorescence dyeo 2) indirect immunofluorescence: staining in which a secondary (which carries the fluorophore, recognises the primary antibody and binds

to it.) antibody labeled with fluorochrome is used to recognize a primary (recognizes the target molecule and binds to it) antibody. Advantages of indirect:

(1). Gives an amplification effect -- more tag or label ('signal') per molecule of target protein. (2). Requires only one labeled antibody to identify many proteins. Same labeled secondary antibody can be used to bind to ("light up") many different proteins. (Preparation of labeled antibody is difficult and expensive.)

Autoantibodies are detected on specific substrates• Anti-dsDNA Ab Crithedia Lucilae substrate• ANA on Hep-2 substate on mouse stomach kidney substrate• APA. ASMA(CT2), AMA. – on mouse stomach kidney

substrate• Anti LKM on mouse liver stomach kidney (CT3)• ANCA on neutrophil substrate• Antithyroid Abs on Thyroid tissue

STAINING PATTERNS Diffuse / homogeneous: antibodies to histone Rim: antibodies to nuclear envelope proteins and to double-stranded (ds) DNA Speckled: antibodies to Sm, RNP, Ro/SS-A, La/SS-B, and other antigens Nucleolar: associated with diffuse scleroderma Centromeric: highly specific for the CREST syndrome

Deficiencies in the Classical Pathway: C1q, C1r, C1s, C4, C2, C1-Inh Primary deficiency of C1q, C1r, C1s or C4 is closely linked to development of systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA)

The Centers for Disease Control and Prevention (CDC) recommend ordering several tests to help determine whether a person is susceptible to EBV or to detect a recent infection or a prior infection, or a reactivated EBV infection. These tests include: Viral capsid antigen (VCA)-IgM, VCA-IgG, D early antigen (EA-D), Epstein Barr nuclear antigen (EBNA)

Adsorptions - a method designed to remove antibody from serum by incubating it with red cells positive for the corresponding antigen.Applications1. Removing autoantibody activity to permit detection of possible coexisting alloantibodies. For example, if a patient has an autoanti-1, an autoadsorption at

4°C can be done to remove it, and the autoadsorbed serum can be run against a panel to identify any possible alloantibodies present.2. Reagent preparation: removing anti-A or anti-B or other unwanted antibodies from serum that contains an antibody suitable for reagent use, e.g., serum

from a group A donor containing anti-D can be adsorbed with group B Rh(D) negative blood cells: the anti-B will be adsorbed, but the anti-D will not.

3. Separating multiple antibodies to aid in identification: for example, if you suspect a serum contains anti-c and other unidentified antibodies that react with R1R1 cells, you could adsorb the serum with R1R1 cells. Anti-c would remain free in the supernatant and could be confirmed by testing the supernatant with panel cells. The antibodies which attached to the R1R1 cells could be eluted and identified.Note: adsorptions are often done in conjunction with elutions

4. Confirmation of the presence of a weak antigen on red cells: this can be done by proving their ability to adsorb and remove specific antibody. For example, supposing a patient's ABO group gave the following results:

anti-A anti-B A1 cells B cells -- -- -- 4+

The patient may be a group A with a very weak A antigen (weak subgroup of A). His red cells could be incubated with anti-A at 4°C for an increased time (e.g., 4 - 12 hours) and then an elution could be prepared. If the eluate contain anti-A, it is possible to conclude the A antigen must have been on the red cells.5. Confirmation of antibody specificity: if you suspect a serum contains an antibody (e.g., anti-c) plus one or more antibodies (e.g., anti-K and anti-Fya), you can

confirm the anti-c by doing an adsorption followed by an elution, e.g., incubate serum with red cells that are Fya-, K-, but c positive, then do an elution of the adsorbed cells and test it with panel cells to confirm it contains anti-c.

Elution- This type of elution is a method mainly used to release antibody molecules from the red cell membrane (when tests show the DAT is positive) for the purposes of identifying the antibody.Applications1. Investigation of HDN: an elution is done on the infant's red cells, and the resulting eluate is tested for the presence of the mother's IgG antibody which has

crossed the placenta and sensitized fetal cells.2. Investigation of IAHA (warm, cold, and drug-related types): an elution is done on the patient's red cells and the resulting eluate is tested for autoantibody

specificity.3. Investigation of hemolytic transfusion reactions: an elution is done on the patient's post-transfusion red cells, and the resulting eluate tested for antibody

specificity. Note that in this case, even though the antibody elutes from the patient's red cells, it is not an autoantibody as it actually eluted from the donor's red cells now in the patient's circulation.

4. Separation of multiple antibodies to aid identification.5. Confirmation of antibody specificity.In cases of HDN, AIHA, and hemolytic transfusion reactions it is necessary to elute the antibody sensitizing the patient's red cells and to identify it by testing the eluate against a panel. There are numerous methods available for this type of elution, including Rubin's diethyl ether, Landsteiner heat at 56°C, chloroform, xylene, and acid elutions. These methods cause intact antibody to be released from the red cells either by disrupting the antigens, or by creating conditions that favour dissociation of antibody from antigen, although the exact mechanisms by which they elute antibody are not clear.