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LABORATORY DIAGNOSIS OF VIRAL DISEASES
Learning Objectives: โ At the end of the session, the students must be able to: โ Describe the general considerations in viral specimen collection. โ Discuss the different techniques in virus isolation and identification. SETTING UP A CLINICAL VIROLOGY LABORATORY SPECIMEN COLLECTION General Principles: โ Specimen collection depends on: REMEMBER โ specific disease syndrome โ viral etiologies suspected โ time of year Note: etiology- origin of disease โ Specimen type and viruses suspected should be indicated in the requisition slip Note: indicate the type of specimen and suspected virus also notify if there is a presence of a RARE agent is
present viruses like SARS-COV 2, Influenza, H1N โ Specimens for detection of virus should be collected ASAP following the onset of symptomatic disease. โ Virus may no longer present as early as 2 (two) days after appearance of symptoms SPECIMENS FOR THE DIAGNOSIS OF VIRAL DISEASES DISEASE CATEGORIES & PROBABLE VIRAL AGENT
SEASON OF COMMON
OCCURRENCE
SPECIMEN OF CHOICE
RESPIRATORY
Adenoviruses Year - round Throat / nasopharynx Influenza virus Winter Throat / nasopharynx Parainfluenza Year - round Throat / nasopharynx Respiratory syncytial virus Winter Throat / nasopharynx SARS Coronavirus Winter Throat / nasopharynx Metapneumovirus Winter Throat / nasopharynx Rhinoviruses (common colds) Year - round Nasal Sin nombre virus Spring, Summer Serum DISEASE CATEGORIES & PROBABLE VIRAL AGENT SEASON OF COMMON OCCURRENCE SPECIMEN OF CHOICE OTHERS DERMATOLOGICAL AND MUCOUS MEMBRANE Enterovirus Summer, Fall Stool, throat / nasopharynx Vesicle fluid or scrapings Herpes simplex Year - round Vesicle fluid or scrapings
CONGENITAL AND PERINATAL Note: present from or at birth Enterovirus Summer, Fall Throat/Nasopharynx, CSF, urine Rubella Year - round Urine Serum for Ab detection Cytomegalovirus Year - round Urine Serum for Ab (IgM) detection Herpes simplex virus Year - round Vesicle fluid Parvovirus Year - round Amniotic fluid, liver tissue Rubella Year - round Urine Serum for Ab (IgM) detection DISEASE CATEGORIES & PROBABLE VIRAL AGENT SEASON OF COMMON OCCURRENCE SPECIMEN OF CHOICE OTHERS GASTROINTESTINAL DISEASE STOOL Adenoviruses (Serotypes 40-41) Year - round Stool (EIA or EM) Noroviruses Summer Stool (EIM) Rotavirus Winter, spring Stool (EIA, Latex) EYE (OCULAR DISEASE) Adenoviruses Year - round Throat/nasopharynx Conjunctival swab or scrapings Herpes simplex virus Year - round Conjunctival swab or scrapings Varicella -zoster virus Year - round Conjunctival swab or scrapings DISEASE CATEGORIES & PROBABLE VIRAL AGENT SEASON OF COMMON OCCURRENCE SPECIMEN OF CHOICE OTHERS POST TRANSPLANTATION SYNDROME Note: virus that can be acquired in organ transplantation Cytomegalovirus Year - round Urine Stool (EIA or EM)
Epstein-Barr virus Year - round Stool (EIM) Human herpesvirus-6 Year - round Stool (EIA, Latex) Herpes simplex Year - round MYOCARDITIS, PERICARDITIS AND PLEURODYNIA Note: sudden occurrence of resonating chest pain Coxsackie B Summer, fall Throat/nasopharynx Pericardial fluid Hemorrhagic fevers Ebola/Marburg viruses Year - round Tissue, respiratory secretions, serum Lassa fever virus Throat/nasopharynx Urine, serum Ab detection Note: Maโam Dianne did not emphasize on the table but itโs our responsibility nalang daw to read and understand -- included in the quizzes and exams :) Collection of Common Specimens I. Throat, Nasopharyngeal Swab or Aspirate โ Nasopharyngeal aspirates โ superior in general; Note: pinaka pinipili nila sa lahat ng specimen for respiratory diseases because it could recover MOST of the viruses โ Throat swab โ considerably more convenient; collected by the MT Note: acceptable in recovering enteroviruses, adenoviruses, and HSV โ Nasopharyngeal swab or aspirate โ detection of RSV, influenza and parainfluenza โ Nasal spx - Rhinovirus II. Bronchial and Bronchoalveolar Washes โ Bronchoscopy โ wash and lavage fluid collected Note: the medical practitioner will locate the lower respiratory tract
โ Cerebrospinal fluid (CSF), pericardial and pleural fluids โ Enteroviruses, HSV, VZV, influenza viruses or CMV Note: not done by medical technologist but by medical practitioner VII. Blood โ Viral culture of blood โ primarily to detect CMV โ HSV, VZV, enteroviruses and adenovirus โ occasionally encountered โ 5-10 mL of anticoagulated blood (heparin, citrate or EDTA) โ CMV detection โ Citrated blood โ other viruses VIII. Bone Marrow โ Sterile tube w/ anticoagulant โ Heparin, citrate or EDTA โ Parvovirus B โ Specimen collected by aspiration Note: not done by medical technologist but by medical practitioner IX. Tissue โ Tissue specimens โ useful for detecting viruses infecting the lung (CMV, influenza virus, adenovirus, sin nombre virus); brain (HSV) and gastrointestinal tract (CMV) โ Collected through surgical procedures X. Serum for Antibody Testing โ Acute and convalescent serum specimen โ detect antibody to specific viruses โ Acute specimen โ collected ASAP after appearance of symptoms Note: acute - sudden onset of symptoms โ Convalescent specimen โ collected a minimum of 2-3 weeks after the acute specimen Note: convalescent - gradual recovery from the illness they collect again a convalescent specimen โ 3 to 5 ml of serum - venipuncture
Specimen Transport and Storage โ All specimens collected should be processed by the lab. immediately. โ Specimens should not be allowed to stand at room or high temperature. Should be placed on ice or if there is a delay, it should be refrigerated NOT frozen. โ Specimens should be placed on ice and transported to the lab immediately. โ If delays occur, refrigerate specimens, not frozen prior to processing. โ Processing of spx. should be done within 12 โ 24 hours after collection. โ Under unusual circumstances, spx. must stored first before processing: โ storage up to 5 days โ spx. shld. be stored at 4ยฐC โ for 6 or more days โ (-20ยฐC) โ (-70ยฐC) โ spx. subjected to freezing shld. be diluted or emulsified in viral transport medium โ For commercial kits, spx. for molecular testing should be stored and transported accdg. to the manufacturerโs instructions Ultra freezer: -20 to -70 C collection bag: place refrigerant or ice โ Swabs spx. (rayon & dacron) โ be emulsified in a viral transport medium before transporting to the lab. โ Viral transport media โ used to transport small volumes of fluid spx., small tissues & scrapings and swab spx. REMEMBER: โ composed of Buffered saline, proteins (serum, albumin, gelatin) - stabilize virus and antimicrobials (penicillin, streptomycin, vancomycin, gentamicin, amphotericin) to prevent overgrowth of bacteria and fungi โ Transport media : Stuartโs medium, Annieโs medium, Leibovitz-Emory medium, Hankโs balanced salt soln. (HBSS), Eagleโs tissue culture โ Blood viral culture โ transported in a sterile tube w/ anticoagulant and must be kept refrigerated until processing โ Blood spx. For viral serology โ transported in a sterile tube, serum must be separated from the clot ASAP
Note: for example the patient has several specimens hindi pwedeng i open naten ng sabay sabay ang mga specimens na ito or kahit from other patient hindi pwedeng sabay sabay PROCESSING is done ONE AT A TIME
- Fluid spx. considered to be sterile; can be inoculated directly to the cell culture.
- fluid specimens in a viral transport media should be vortexed before inoculation; to break up virus-containing cells and resuspend the inoculum - sterile glass beads
- Blood viral cultures requires special processing to isolate leukocytes (WBC) - inoculated to cell culture tubes Rapid Shell Viral Cell Cultures โ commonly used to detect viruses Virus Detection Methods I. Cytology and Histology
- Presence of characteristic viral inclusions
- involves the study of the morphologic study of cells or tissues โ Viral inclusions โ intracellular structures formed by aggregates of virus or viral components w/in an infected or abnormal accumulations of cellular materials resulting from viral-induced metabolic disruption; single or syncytial cells โ Syncytial cells โ aggregates of cells fused to form one larger cells with multiple nuclei โ Cytology โ PAPโs smear or Giemsa-stained cytologic smears; most frequently used to detect infections with VSZ (Varicella-Zoster) and HSV (Herpes Simplex Viruses) โ Tzanck test โ stained smear of cells from a base of skin vesicle (cytology) โ Histologic Examination โ CMV, adenovirus, parvovirus, papilloma virus, molluscum contagiosum virus; Histologic slides are stained with H&E (Hematoxylin & Eosin Stain), and PAPs smear Cytology and Histology โ less sensitive; not preferred in virus detection. Helpful in detecting viruses w/c are difficult or dangerous to isolate or dangerous to cultivate (ex: parvovirus, rabies virus) Cytology smear of Molluscum contagiosum virus - There is an aggregate of cells, meaning buo-buo or dikit-dikit yung mga cells, forming into a large cell with multiple nuclei. The nucleus are stained dark. II. Electron Microscopy โ Labor intensive and relatively insensitive
โ Most helpful for detection of viruses that do not grow readily in cell cultures โ Works best if the titer of virus is at least 10^6 โ 10^7 particles/ml. โ Allows easy visualization of virus particles present in numbers that are too small for easy direct detection. It could identify viruses that are very small. โ Addition of specific antiserum โ virus particles to form Antibody-bound aggregates โ more easily detected than single virus particle โ Most useful detection for gastroenteritis viruses; (norovirus, coronavirus, astroviruses) โ Viruses causing encephalitis (HSV, measle virus and JC polyomavirus) โ Etiology of newly recognized viral syndromes: โ Ebola virus โ 1970, Africa โ Sin nombre virus โ 1990, US ** CORONAVIRUS** EBOLA VIRUS III. Immunodiagnosis (Antigen Detection) โ Viral antibody reagents: fluorescent antibody, enzyme immunoassay, radioimmunoassay, latex agglutination, immunoperoxidase test โ detect viral antigens III. A. Direct and Indirect Immunofluorescence Testing โ Direct immunofluorescence testing โ use of a labeled antiviral Ab called fluorescein isothiocyanate (FITC) ; rapid and specific but less sensitive; best when a large quantity of virus is suspected example is the detection of respiratory syncytial virus (RSV). โ Indirect immunofluorescent - 2-step test; unlabeled antiviral Ab added to the slide followed by FITC; increased sensitivity; could be used for low quantities of viruses.
III. B. Enzyme-linked immunosorbent assay (ELISA) โ Solid phase ELISA โ small test tube or microtiter tray; detection of rotavirus in stool spx. โ Membrane-bound ELISA โ low-volume testing ; rapid results are needed (less than 30 minutes); handheld reaction chamber w/ a cellulose membrane ; chromogenic reaction (color reaction) โ RSV & Rotavirus (solid-phase) โ Influenza Virus (membrane-bound) Advantages of enzyme immunoassays: โ Use of nonradioactive, relatively stable reagents โ Results can be interpreted qualitatively (positive or negative) and quantitatively (titer or degree of positive reaction) โ Sensitive and simple to perform, can be easily automated โ Important note: have frequently indeterminate or borderline interpretative category โ low levels of viral antigen or background interference III. C. Radioimmunoassay (RIA), Immunoperoxidase & Latex agglutination โ Radioimmunoassay โ replaced by ELISA because it uses expensive equipment and disposal procedures โ has issues in disposal procedures due to radioactive materials. โ Immunoperoxidase staining โ commonly used to stain histologic sections for virus; less popular than
immunofluorescent staining โ Latex agglutination โ easy and inexpensive method; lacks sensitivity IV. Enzyme-Linked Virus Inducible System (ELVIS) โ Uses a BHK (baby hamster kidney) cell culture system w/ a cloned beta-galactosidase gene โ expressed only when cells are infected with a virus โ Enzyme-linked Virus Inducible System (ELVIS-HSV test system) โ genetically engineered BHK cells - sold in multi-welled microtiter plates โ After incubation overnight, growth of HSV result in the production of beta galactosidase enzyme by genetically engineered baby hamster kidney (BHK) cells โ Beta galactosidase - serves as โreporterโ molecule โ Positive staining โ presence of HSV-1 or HSV- โ Herpes simplex virus - the one that causes sexually transmitted disease. V. Molecular Detection using Nucleic acid Probes and Polymerase Chain Reaction (PCR) Assays V. A. Nucleic acid detection โ nucleic acid probes (short segments of DNA that hybridize w/ complementary viral DNA or RNA segments); used if viral culture is not possible โ Probe labeled w/ a fluorescent, chromogenic, radioactive tag โ allow detection if hybridization occurs โ DNA probe โ detect papillomavirus DNA โ Most useful when amount of virus is relatively abundant; viral culture โ not possible; immunoassay lacks sensitivity or specificity V. B. Polymerase Chain Reaction (PCR) โ method which duplicates short DNA targets thousands to million-fold โ โ amplicons โ โ readily ID presence of a specific virus PCR reaction โ automated and very rapid โ Real-time PCR โ rapid testing; DNA viruses โ RT-PCR (Reverse Transcriptase) โamplify and detect RNA viruses using the enzyme RT; used in SARS-CoV-2 (RNA virus) ** viruses are divided into two: DNA and RNA viruses REAL-TIME POLYMERASE CHAIN REACTION (PCR) OR REAL-TIME RT-PCR โ CNAT: Quantitative Real-time PCR assay for cytomegalovirus (CMV) โ FLUNAT: influenza A and influenza B, influenza A subtype identification (i.e. influenza A of swine origin 2009, seasonal H1 or H3 influenza A)
โ During the cell culture there is a production of CO2 & acidification of growth liquid or the bicarbonate buffering system โ Incubation occurs for about 1-4 weeks ** the cell culture tubes are periodically inspected microscopically for the presence of virus - Cytopathic effect (CPE) or the presence of dying cells (monolayer cells?) in the cell culture tube CYTOPATHIC EFFECT (CPE) TEST DETAILS โ ISOLATION OF VIRUSES IN TUBE CULTURES (VIRC) โ Respiratory syncytial virus (RSV) โ Enteroviruses โ Coxsackie virus (A and B) โ orthomyxoviruses โ herpes viruses (CMV, HSV1, HSV2, VZV) (influenza A and B) โ parainfluenza types 1, 2 and 3 โ Adenovirus The above pictures show the CPE of vero cells. The virus had caused holes to form in the vero cells monolayer as the cell membranes have been destroyed. Some of the cells have detached from the monolayer. More cells will be infected if they are incubated for a longer period, this will result in more cells to die and more holes to be formed in the cell monolayer. Conventional Cell Culture โ Two kinds of media:
โ Growth medium โ serum-rich nutrient medium โ Support rapid cell growth โ โ feeding โ โ removal of old medium following by addition of fresh medium โ Maintenance medium โ similar w/growth medium but less serum โ used to keep cells in steady state for metabolism โ Both are prepared w/ Eagleโs minimum essential medium (EMEM) in Earleโs balanced salt solution (EBSS); include antimicrobials to prevent overgrowth of fungi and bacteria โ Cell line โ a cell culture that has been passed or subcultured in vitro Types of cell lines: โ Primary cell lines โ passed only once or twice; โ Ex. primary monkey kidney cells โ Low passage cell lines โ remains virus-sensitive (20-50 passage); โ Ex. lung fibroblast (human diploid fibroblast) - commonly used โ Continuous cell lines - can be passed and remained sensitive to virus infection indefinitely โ Hep-2 cells (human epidermoid carcinoma cells) โ โBlind passageโ - passing of the cells from one tube to another; used to detect viruses that may not produce CPE in cell culture tube
2. Shell Vial Cell Culture / Rapid Shell Vial Cell Culture - rapid modification of conventional cell culture; virus detected more quickly (w/in 24 hrs.) โ difference from the conventional cell culture is the addition of round coverslip at the bottom; coverslip is stained w/ virus specific immunofluorescent conjugates โ fluorescent inclusions โ confirm presence of virus โ CMV, VZV (viruses with long incubation before CPE production) VII. Viral Serology โ Used to detect immune status and make diagnosis of infections when virus cannot be cultivated in cell culture or detected readily by immunoassay or molecular assays Serological Methods:
