Viral Specimens Selection and Collection PDF
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Dr.Sheylan S. Abdullah
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This document provides information about viral specimen selection and collection for diagnostic purposes. It covers different sample sites, associated viral agents, and specimen transport methods.
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Lab 1 / Virology /7th Semester / Dept. of (MLT) _ Dr.Sheylan S. Abdullah Viral Specimens Selection and Collection: Specimen Processing for Diagnosing Viral Diseases 1. Specimen selection depends on the specific disease syndrome, viral etiologies suspected and time of year. 2. Viruses...
Lab 1 / Virology /7th Semester / Dept. of (MLT) _ Dr.Sheylan S. Abdullah Viral Specimens Selection and Collection: Specimen Processing for Diagnosing Viral Diseases 1. Specimen selection depends on the specific disease syndrome, viral etiologies suspected and time of year. 2. Viruses are in highest concentrations during the first several days following onset of symptoms. Therefore, samples should be collected early in the disease course. 3. Samples should generally come from the infected site. Sample sites and associated viral agents a. Skin infections: Cutaneous infections often result in a rash that, depending on the virus, can have a variety of presentations. Sufficient number of materials collected from lesion can be done by swabbing. when samples taken from skin, always scrape the epithelium because the viruses are intracellular parasites, don't pick up just mucus or secretions. If blisters were founded, open these blisters and pick up the materials from deep lesion, then put the swab in the Glycerol media and send it to the lab. Cutaneous Infections caused by HSV-1, HSV-2, VZV, echovirus, measles virus, rubella virus, enterovirus and parvovirus B-19. b. Respiratory infections: Sputum or throat swabs Upper respiratory tract infections are commonly caused by viruses, 1 including rhinovirus, influenza virus, parainfluenza virus, respiratory Page syncytial virus (RSV), Epstein-Barr virus (EBV), and coronavirus. Croup and bronchitis can be caused by influenza virus, parainfluenza virus, RSV, and adenovirus. Pneumonia in children can be caused by RSV, parainfluenza virus, adenovirus, and varicella-zoster virus (VZV). Pneumonia in adults can be caused by influenza virus, VZV, cytomegalovirus (CMV), and RSV. Lab 1 / Virology /7th Semester / Dept. of (MLT) _ Dr.Sheylan S. Abdullah c. Central nervous system For diagnosis of meningitis, cerebrospinal fluid (CSF) and serum, as well as stool or throat swabs, can be collected because viruses are sometimes shed into these sites. enterovirus, echovirus, herpes simplex virus type 1 (HSV-1), HSV-2, and VZV. In cases of encephalitis, brain biopsy material and sometimes serum are used. caused by HSV, VZV, and arboviruses. d. Urogenital infections: Needle aspirates and endocervical and urethral swabs Genital tract infections are typically sexually transmitted by HSV-2 and human papillomavirus. CMV, mumps, rubella, measles, polyomaviruses and adenoviruses can be detected in urine. e. Gastrointestinal tract: Stool samples and rectal swabs Including rotaviruses, adenoviruses and calciviruses. f. Eye infections: Eye swabs and corneal scrapings Infections caused by HSV, adenovirus, and VZV. g. Biopsy and autopsy: My be taken by needles or knife. Swab For diagnosis of viral infections, swabs should be: made of rayon, should not be made of cotton. Swab’s shaft should be: made of plastics or metal, should not be made of wood. 2 Specimen transport and storage: Page 1. All specimens for detection of viruses should be collected with aseptic technique and transported immediately to the laboratory by using transport media (in screw capped tube) and should be placed in ice, specimens for viral isolation should not be kept at room or higher temperature. Under unusual circumstances, specimens may need to be held for days before processing. For storage up to 5 days, hold specimen at 4°C. Storage for 6 or more days should be at – 20°C or preferably at – 70°C. Lab 1 / Virology /7th Semester / Dept. of (MLT) _ Dr.Sheylan S. Abdullah 2. Some antibiotics (e.g penicillin, streptomycin, nystatin) should be added to viral transport media especially when contamination with microbial flora is expected. Examples of successful transport media include Stuart’s medium, Amie’s medium and Hank’s solution. Viral transport media (VTM) is used to: Preserve viral infectivity within the specimen Prevent specimen from drying Stop the growth of bacteria and fungi VTM contains: - Saline (adequate ion concentration) - Proteins (albumin or gelatin) - buffer (adequate pH) - Antibiotics and fungicides 3. Stock of Glycerol medium: Glycerol medium is an isotonic buffer solution contain minerals, albumin, gelatin, yeast extract and antibiotics. This medium is useful for: a- prevent drying out of the samples. b- inhibition of bacterial growth c- diluting antibodies which may come with the samples. 4. Centrifuge under refrigeration, the supernatant will contain the viruses. 5. Filter the supernatant to give crude liquid of viruses. 6. Detection of viruses in this crude liquid of viruses by 3 methods It will be mentioned in the later lectures. Laboratory Diagnosis of Viral Infections Diagnostic virology is concerned with identifying the virus associated with clinical signs and symptoms. Procedures most commonly used include: 1. Detection of a meaningful immune response to the virus (antibody or cell- mediated) by immunologic assay(s) 3 2. Identification of the agent by staining of specimens or sections of tissue (light Page and electron microscopy) 3. Isolation and identification of the agent (cell culture or fertile eggs) 4. Detection of viral nucleic acid (probes or amplification). Lab 1 / Virology /7th Semester / Dept. of (MLT) _ Dr.Sheylan S. Abdullah 1. Detection of Immune Response Detection of rising titers of antibody between acute and convalescent stages of infection, or the detection of IgM in primary infection Often, 1 it is difficult to identify a virus in relation to the disease observed, or when 2 conducting a retrospective study of a population to determine exposure to a virus, or when 3 measuring the response of an individual to a vaccine. In these cases, indirect methods of measure are needed, such as measuring antibody response to the virus of interest. Several methods exist for this purpose. A few of the most commonly used methods include Newer Techniques Classical Techniques 1. Radioimmunoassay (RIA) 1. Complement fixation tests (CFT) 2. Enzyme linked immunosorbent assay 2. Haemagglutination inhibition tests (EIA) 3. Particle agglutination 3. Immunofluorescence techniques (IF) 4. Western Blot (WB) 4.Neutralization tests The principles of these assays are fundamentally the same, they depend upon antibody-antigen interactions and consist of a known virus or viral protein, a patient sample (usually serum), and an indicator. If antibodies are present in the patient’s serum, they will bind to the virus. If no antibodies are present, no binding will occur. The indicator is observed to determine whether the sample is positive or negative for antibodies. 2. Microscopy It includes: Light Microscopy (L.M.) has been traditionally used in directly demonstrating viral infections by detecting the viral inclusion body in smear & tissue. Inclusion bodies are dense aggregates of stainable substances, usually proteins. They can be either intra nuclear (present inside the nucleus) of 4 infected well or intracytoplasmic (present inside the cytoplasm of the Page infected cell). Viruses that are assembled in the nucleus (usually DNA viruses) like herpes simplex viruses, Varicella zoster virus, Cytomegalo virus & adeno virus forms intra nucleus inclusions. While viruses that are assembled in the cytoplasm (usually RNA viruses) like Respiratory syncytial virus, rabies virus & viruses of the pox group forms intra cytoplasmic inclusion bodies. Lab 1 / Virology /7th Semester / Dept. of (MLT) _ Dr.Sheylan S. Abdullah The intracytoplasmic lesions of rabies virus are known as Negri bodies, intracytoplasmic inclusions of the pox viruses known as Guarneri bodies. Electron microscope (E.M.): more useful for seeing structure of viruses, symmetry and No. of capsomeres and used in the study of clinical specimens & cell cultures. Electron microscope has been used effectively in the detection of viral agent of gastro enteritis especially those that are not recovered by conventional cell cultures. Electron microscopes are useful in ultra- structural observations especially for research purposes. Flourescence microscope (F.M): used for detection Flourescent Ab 3. Molecular methods Molecular methods like polymerase chain Reaction (PCR), Real time polymerase reaction (RT-PCR), Nucleic acid. involves the detection of viral DNA or RNA sequences in nucleic acid extracted from specimens. It is highly sensitive and specific and is a popular technique for identifying adenovirus in nasopharyngeal washings, CMV in urine, and HIV in the blood of seronegative individuals. They are fast becoming routine diagnostic methods in developed countries. 7/Oct./2024 5 Page - To identify the virus associated with clinical signs and symptoms - To measure the response of an individual to a vaccine - To determine exposure to a virus in a population