Parasitology and Serological Testing for Viruses PDF

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Summary

- A parasite is an organism that lives on or in a host at the hosts expense. - Definitive Host, where the organism goes through its sexual multiplication phases. - Intermediate Host, where the larvae and asexual stages are found. - Parasites typically exhibit food and water transmission.Unicellular ...

Description

MLSC-3131U, Clinical Microbiology II -

A parasite is an organism that lives on or in a host at the hosts expense. Definitive Host, where the organism goes through its sexual multiplication phases. Intermediate Host, where the larvae and asexual stages are found. Parasites typically exhibit food and water transmission.

Unicellular Protozoans -

Amebae, asexual reproducing and move by pseudopodia. Have Trophozoite and cyst stages. Flagellates, move by flagella, undergo asexual reproduction, and have Trophozoite and cyst stages. Ciliates, move by cilia, have Trophozoite and cyst stages, and undergo asexual reproduction. Sporozoans, classified by their complex reproductive cycle. May undergo both asexual and sexual reproduction.

Multicellular Helminths -

Roundworms, have adult male and female roundworms. They have egg, larvae, and adult life stages. May be free living or require intermediate hosts. Flatworms o Tapeworms, very ribbon like hermaphrodite organisms. Have egg, larvae and adult life cycles. Use mammals and insects as intermediate hosts. o Flukes, mostly hermaphroditic. Have an egg, miracidium, cercaria and adult life cycle stages. Use fish, snails, and crabs as their intermediate hosts.

Protozoans -

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Typically have two stages (Trophozoite and cyst). Some have only a Trophozoite stage. Trophozoites are motile, replicate and feed while cysts are immobile, resistant, infective and do not reproduce. Most live in the intestinal tract as Trophozoites and are transferred in the cyst form. Few amebae are free living Trophozoites and enter the body directly through mucosal membranes as Trophozoites. Protozoans are identified by: o Life Stages, some may or may not have both the Trophozoite and cyst stage. o Size o Number of Nuclei, or nuclear structure. The treatment is Metronidazole.

MLSC-3131U, Clinical Microbiology II Amoeba -

Entamoeba histolytica, an important pathogenic ameba. Ingested as a cyst and becomes a Trophozoite in the intestine where it undergoes asexual reproduction in the colon. If a Trophozoite is passed through the stool it will die due to the harsh environment. Causes hepatic abscesses. Naegleri fowleri, free-living brain eating amoeba. Acquired from warm waters in the USA. Must penetrate the sinuses where it will travel through the olfactory nerve to the brain. Infection only causes infection if water is forced up the nose. Typically diagnosed in an autopsy by the presence of Trophozoites in the brain. Acanthamoeba keratitis, cause serious infections of the cornea. Mostly occur in people who use contact lenses. Rinsing contacts in tap water, failure to disinfect lenses or wearing lenses while swimming are risk factors for infection.

Flagellates -

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Giardia lamblia, pathogenic with a worldwide distribution. Transmitted through contaminated drinking water that contains cysts. Causes gastroenteritis and travelers diarrhea. The Trophozoite shows falling leaf motility. Trichomonas vaginalis, pathogenic and causes STI. Tendency to be associated with gonorrhea. Causes purulent vaginal discharge in females and thin urethral discharge in males. Cultured on Diamond’s media and does not have a cyst stage.

Sporozoans -

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Have complex life cycles that can involve asexual (intermediate host) and sexual (definitive host) reproduction. Sexual cycles occur in definitive hosts while asexual cycles occur in intermediate hosts. Toxoplasma gondii, affects fetuses severely. Can be asymptomatic or mild and mimics mononucleosis. The immunocompromised and congenital patients are at risk the most. Transmitted through ingesting the Oocyst from a definitive hosts fecal/oral route (usually cats). Shows neonatal transfer through transplacental passage. Also transmitted through organ transplants. Cryptosporidium parvum, human to human zoonosis transmission. Very resistant to chlorination and temperature. The Oocysts are thick walled and spherical, so it is also acid-fast staining.

Helminths Have complex life cycles involving two or more hosts. Adult worms produce eggs in the definitive host and the eggs mature in the environment or other species. Flat Worms -

MLSC-3131U, Clinical Microbiology II

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Flukes, dorsoventrally flat and hermaphroditic. They have leaf-like shapes and their eggs have an operculum, which is a hatch allowing the larvae to escape. o Blood Flukes, include Schistosoma spp. These are different from other flukes. Schistosoma are cylindrically shaped, lack operculum and have a spike on one end. Transmitted through skin penetration from contaminated water while swimming and causes swimmer’s itch. Tapeworms, also known as cestodes long ribbon-like bodies. Have segments called proglottids, with each one having both male and female reproductive organs. Proglottids are sloughed off as it grows to cause further infection. The segments closest to the head have the least mature sexual organs while those towards the end of the worm are more fully developed. o Diphyllobothrium latum, usually found in fish and are ingested in pickled/raw freshwater fish. Have an operculum on their eggs. o Taenia saginata, beef tapeworm. o Taenia soliunm, pork tapeworm. Laboratory identification is usually done using the proglottids or visually through the egg size, colour, and presence of operculum.

Roundworms Enterobius vermicularis, common in children 5-10 years old. Female lays eggs on the perianal area, causing itching in that area. Spread through bad hand hygiene in children when they scratch the perianal area and eat food without washing. Eggs colourless, easily seen under 10x, oval, thick shelled and flattened on one side. The light from the microscope will cause the larvae to move inside the egg. - Trichuris trichura, also known as whipworm. Ovum are found in stool and the worm looks like a whip (thicker and one end than the other). - Ascaris lumbricoides, intestinal roundworm. The female is larger than the male and these are the largest of the roundworms. The eggs are round and bumpy on the edge. Patients can ingest a fertilized egg which will hatch in the colon. The larvae then cross the colon intestinal wall to migrate up to the trachea and the lungs. - Hookworms, caused by Necator americanus (New World hookworm) and Ancylostoma duodenale (Old World hookworm). - Strongyloides stercoralis, very clinically significant organism. Very small and thread like. Transmission through infected soil. Patients can be asymptomatic, but when placed on chemotherapy the disease starts to affect every major organ. - Diagnosed by checking for anti-strongyloides antibodies routinely. - Because the organism affects the lungs, they may be present in sputum samples. If inoculated onto agar media, the bacteria covered worms will move around on the plate. This will leave behind a trail of colonies tracing where the roundworm has been. - Squiggly bacterial growth is evidence on Strongyloides infection. The larvae are also seen in the sputum gram stain. Parasitology Specimen Collection and Examination -

MLSC-3131U, Clinical Microbiology II

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Preserved Fecal Specimens, the sample of choice for intestinal parasites and comes in SAF fixative. Fresh stool is not ideal but accepted if it is examined within an hour from collection. OMP, stool for ovum and parasite. Uses formalin fixative to fix the parasite to avoid disintegration. From that we do stains, wet mounts, and concentrates. Three specimens are collected, spread out over 10 days with no collections on the same day. Specimens should not be contaminated urine or water and are rejected if they are contaminated. Stool specimens are examined macroscopically for intact worms. The motile proglottids from a tapeworm can sometimes be seen. Direct Wet Mount, some stool is placed on a slide, cover slipped and examined for Trophozoites and helminth eggs under a microscope. Not ideal since the one drop of stool is not representative. Used to view helminth eggs and large trophozoites. A small amount of fecal specimen is emulsified into a drop of iodine solution or saline. Overlayed with a coverslip and sealed with nail polish. The iodine will highlight internal parasite structures, destroy trophozoite motility and stain glycogen. Direct wet mounts are examined with low power and the high-power is used to identify characteristics. o Advantages, may detect parasites that do not concentrate well. o Disadvantages, eggs, cysts and Trophozoites can be low in number or confused with fecal debris. Concentrate, used to look for protozoan cysts, oocyts, helminths eggs and larvae. Separates parasites from fecal debris and increases the chances of detection when these are in low numbers. Divided into sedimentation techniques and flotation techniques. o Sedimentation, uses a solution with a lower specific gravity than the parasites, ethyl alcohol to dissolve fats and formalin to fix and preserve. Mixture of broth and specimen is centrifuged to concentrate the parasites in the pellet of the tube. o Flotation, uses a solution with a lower specific gravity than the parasites. Allows the parasite to float to the top of the tube. If the parasite has a very large ovum with a S.G. greater than 1.300 it will sink to the bottom of the tube and be missed. The top layer must be quickly examined to avoid the organism settling at the bottom of the tube over time. Permanent Stains, made from concentrates to provide contrasting colours for both the background debris and parasites present. Provides a detailed organism morphology under oil immersion examination. A smear of the stool is done to look for Trophozoites with the Iron hematoxylin or Trichome stain. Gold standard for ID. Smears are scanned in thick and thin areas at low power from 10x to 40x. Thin areas may be viewed under the 100x, but oil cannot be used to avoid an oil bridge. Takes 1015 minutes. o Iron Hematoxylin, provides a high definition of parasitic structures but has a difficult procedure and significant variability.

MLSC-3131U, Clinical Microbiology II

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o Trichrome Stain, stains the cytoplasm of cysts and Trophozoites blue-green with a tinge of purple. Nuclear chromatin and ingested RBCs appear red-purple and the background stains green. Widely used in the lab. Cellophane Tape Test, used to identify Enterobius vermicularis. Pinworms migrate to the perianal area to deposit eggs overnight. In the morning, a sticky paddle is placed on the surface of the anus in hopes of collecting the eggs on the sticky surface. The paddle is then placed on a slide to be examined microscopically. The CSMLS exam covers parasites that are routinely seen in a microbiology and chemistry lab. These organisms are usually seen in urine microscopy if in the chemistry lab. Trichomonas vaginalis, Enterobius vermicularis, and Schistosoma are commonly seen in the urine because of fecal or vaginal contamination. The ovum of these organisms are transparent and possess a delicate terminal spine. Sputum, Strongyloides, Ascaris lumbricoides and hookworm larvae can be seen on sputum inoculated agar and clinical gram stains.

Quality Assurance in Parasitology -

Quality assurance is done through: o Updated procedural manuals o Controls for stains o Recording of centrifuge calibration o Ocular micrometer calibration o Other materials, including reference books, digital imaged of parasites, permanently stained smears for reference, and external proficiency test programs.

Ectoparasites -

Parasites that live in or on the skin. They receive nourishment through the blood. Include ticks, fleas, bedbugs, lice, and mites.

Serological Testing for Viruses -

Clinical Specimens, can be tested for disease through culturing, genomic, or serological testing for the organism’s antigens. Antibodies cannot be detected in clinical swabs. Serum Specimens, usually used to diagnose disease based on the presence of patient antibodies to the organism. However, some organisms may be detected in serum by both their antibodies and viral antigens.

Acute and Convalescent Antibodies

MLSC-3131U, Clinical Microbiology II

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Primary Response, seen as a small spike in IgM followed by a spike in IgG. Occurs on the first exposure to the organism/antigen. Secondary Response, a quick large spike in IgG is seen followed by IgM. Occurs on the second exposure to the organism/antigen. The memory B cells will remember the previous exposure and be able to produce antibodies quicker during exposure.

Acute and Convalescent Samples -

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An acute and convalescent sample is taken to diagnose patients based on antibody production. Antibody Titre, the highest dilution of the serum where the AB was still detectable. The first sample is taken during the acute phase ASAP. The convalescent sample is usually taken 2 weeks after the acute sample is taken. The acute sample is frozen and will only be tested once the convalescent sample is collected and ready. In an acute sample, usually there is little no IgG or IgM. In a convalescent sample, there will be IgM production and little to no IgG production. This pattern is typically in a recent viral infection. Depending on when the samples are taken, there may be production IgM production without IgG production in the acute sample. Also, the convalescent sample may IgG and IgM or a resolution of IgM, leaving only IgG present (IgM will rise before IgG does in primary exposure). A four-fold rise in the antibody titre indicates a rising titre and is clinically significant and evident of recent infection. A four-fold decrease in the antibody titre is also clinically significant and evident of recent infection.

Determining Patient Immune Status -

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Reactive, patient sample shows evidence of past infection/vaccination. Provides evidence of immunity to the target pathogen. Non-Reactive, patient sample shows no evidence of immunity and indicates susceptibility. Requires re-vaccination. We use antibody detection in serum to determine the patient’s immune status. False Negatives, seen on immunocompromised patients (cannot produce Ab). Lyme disease and Legionnaires Ab can take months to develop. False Positives, seen in patients that have received artificial passive immunity treatment (immunoglobulin administration). Rheumatoid factor also causes cross reactive Ab in autoimmune diseases. Newborns can cause false negatives since they have immature immune systems that will not produce as much Ab. Can also cause false positives in IgG testing (newborns receive IgG from the mother through the placenta. Testing will only reflect the mother’s titre). Newborn serum testing must only look for IgM to indicate a new infection.

Serological Testing to Prevent Congenital Infections

MLSC-3131U, Clinical Microbiology II

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Pre-natal Testing, pregnant mothers are screened for infections that can cross the infection to cause utero-acquired infections in the child. These are usually asymptomatic for the mother but can be detrimental to the baby. TORCH Agents, Toxoplasmosis, Rubella, CMV, and Herpes. Syphilis, HIV, and Hepatitis B infection are also looked for using either antibodies or viral antigens depending on the specimen (clinical swabs are not suitable for Ab detection).

Serological Test Methodologies -

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Direct Agglutination, determines the presence of bacterial antigens directly through agglutination reactions. o Staph Latex, latex particles coated with human fibrinogen and IgG. Causes Staphylococcus with clumping factor or Protein A to cross link and cause agglutination. o Salmonella and Shigella Serology, employs antisera against bacterial cell antigens such as the capsule, flagellum, and cell wall. o Cryptococcal neoformans Agglutination, direct test done on the CSF clinical specimen to detect the organisms antigen using the Ab against the pathogen that are bound to latex particles to cause agglutination. Labelled Immunoassays, include direct/indirect immunofluorescence and the chemiluminescence microparticle immunoassay (CMIA, for syphilis). o Direct Immunofluorescence, detects the organism’s Ag. A nasopharyngeal swab is taken and inoculated onto a slide. A labelled Ab specific for the pathogens antigen is then dropped onto the slide. In a positive reaction, the labelled Ab will bind to the organism and cause fluorescence. Primarily replaced by PCR. The Strep POCT device uses the methodology. o Indirect Immunofluorescence, detects patient Abs to the disease. A well or a slide coated with the organism’s Ag is mixed with patient serum. If there are Abs in the serum against the target pathogen, they will bind but the reaction will not be visible. A labelled anti-human Ab will then be added and will bind to any patient Abs against the disease that are bound to the well. This causes a visible fluorescence in a positive reaction. Negative reactions are seen when no patient AB is present, resulting in no labelled Ab to bind in the well and no fluorescence to be seen. Hepatitis Testing, many hepatitis viruses exist. o Hepatis A, transmitted through the oral-faecal route. o Hepatitis B and C, transmitted sexually or through the blood. A hepatitis panel can be run on a patient to look for Ab production to Hepatitis. This panel looks for anti-HAV IgM in Hepatitis A, and anti-HCV in Hepatitis C. IN Hepatitis B, it looks for anti-HBe (envelope Ab), anti-HBc IgM (core Ab) and anti-HBs (surface antigen Ab). Since Hepatitis B is bloodborne, the panel can also look for viral antigens (both surface and envelope antigens).

MLSC-3131U, Clinical Microbiology II -

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The earliest marker for Hepatitis B infection is the viral surface antigen (HBsAg). This indicates the presence of the virus in the blood. The presence of the core antigen AB (anti-HBc IgM) also indicates acute infection. The presence of AB against the surface antigen (anti-HBs) usually indicates past vaccination. Exposed susceptible individuals can be treated with Hepatitis B Immunoglobulin (HBIG). Incubation Phase, no AB or Ag detected. Acute Infection, surface and envelope antigen are detected with symptoms occurring half way through the phase. Early Recovery, patient mounts an immune response and produces AB against the core, surface antigen and envelope. A spike in IgM is indicative of acute infection. Recovery, shows the presence of anti-HBc, anti-HBs and anti-HBe levelling off. The hepatitis surface and envelope antigen are only present when the patient has the infection. Anti-HBc is only positive in acute or chronic infection. The envelope Abs are present in past infection, chronic infection but not acute infection. Anti-HBs is very important in providing immunity and is the only one present in HBV vaccination....