Laboratory Diagnosis Flashcards
Sample. Once collected, it is recommended to_____ the sample with a_____.
Reliable laboratory results can only be obtained if the specimens are collected, preserved, and transported correctly to laboratory.
emulsify
viral transport medium (VTM)
•Specimen for detection of viruses should be collected as soon as possible after the______ that is when the concentration of the virus is at its highest.
•_____for antibody testing should be collected as early in the disease as possible and also______days later.
appearance of the symptoms
Blood
5 to 10
•_______ specimens (paired sera) are necessary to detect a rise in the titer of the antibodies.
A_____-fold rise in titer between paired sera establishes a positive result.
Two serum
four
In general, specimens for virus isolation should be collected within _____ after onset of illness as virus shedding decreases rapidly after that time.
With only a rare exception, virus cultures are not worthwhile for specimens collected more than____days after the onset of illness.
4 days
7 days
Transport medium, time & temperature
•Except for ________, place all viral specimens in a_______
body fluids (bronchoalveolar lavage, cerebrospinal fluid, urine, blood)
transport medium (UTM or VTM).
- a transport system suitable for collection, transport, preservation, and long-term freeze storage of clinical specimens containing viruses for viral molecular diagnostic testing, including, COVID-19, Chlamydia, Mycoplasma or Ureaplasma organisms.
UTM: Universal transport medium
• Medium formulation inhibits bacterial and fungal growth;
maintains viral viability for 48 hours at room or refrigerated temperature.
UTM
Transport medium, time & temperature
•_______is a buffered medium used to maintain the viability of viruses during their transport to a virology laboratory.
VTM
Viral Transporting Medium
___________is composed of_______ and ______
The solution may be used to wash cells and tissue and to maintain cells in a viable state.
The solution is buffered with phosphate and maintains a physiological pH and osmotic pressure.
Hanks’ Balanced Salt Solution
inorganic salts and supplemented with glucose.
Viral Transporting Medium
is a major component that provides antioxidant, cryoprotectant, and anti-adsorption properties that favor retention of intact virus in solution over lysis and adherence to plastic.
Bovine serum albumin
Viral Transporting Medium
_________ aka_______is a pH indicator frequently used in cell biology laboratories.
To know if sample is still of good quality
Color???
Phenol red (also known as phenolsulfonphthalein or PSP)
Neutral: pink/ red
Viral Transporting Medium
is used to prevent infections caused by a fungus (or yeast).
Nystatin
Viral Transporting Medium
The antibiotics _______ and ______are used to prevent bacterial contamination of VTM due to their effective combined action against gram-positive and gram-negative bacteria.
penicillin and streptomycin
Transport Time and Temperature
•Viruses are unable to survive temperature over _____,_____or____in temperature.
•Viruses can also be damaged be light, drying, change in pH, and bacterial enzymes.
•The usage of_____ will prevent specimens from drying out and help to preserve viral activity.
•All viral specimens should be transported in an_____ with a____
50°C, freezing or fluctuating
VTM
icebox
warning label.
The sooner the specimens reach the laboratory the better.
• Short term storage:
• Long term storage:
+4°C (most viruses stable for 2-3 days)
-70°C or -190°C (LN2) [indefinitely at -70°C]
Viral Sample Information
The following information must be provided:
1. Name, place, age, number of the patient, and details of any recent travel.
2. Type of specimen, if two, mention as the first of second
3.Investigation required.
4.Full clinical information: nature duration, and severity of the disease.
5. Details about immunization and antimicrobial therapy.
6. Name and address of the hospital or health center.
Unacceptable Specimens (10)
- There is discrepancy between the specimen label and the patient name/file number discrepancy in the request form.
- There is no patient name or other unique identifier on the specimen.
- Specimen is too old when received. Excessive delay in transportation.
- There is apparently no specimen in the container.
- The expiration date of the transport medium has been exceeded.
- Inappropriate transport temperature or transport medium.
- Specimen received in a fixative.
- Dried specimen.
- Insufficient quantity.
- Leakage.
Nasopharyngeal secretions
1. Collect a specimen by passing a sterile______ (or rayon with flexible, fine plastic or aluminum shafts), through the floor of the nasal cavity as far as the_____. (________swabs or swabs with wooden shafts are not acceptable.)
- After________, slowly withdraw the swab and immerse the swab in a container of sterile____, cutting off swab wire to allow the bottle cap to be replaced tightly.
3.A specimen of nasopharyngeal secretion can also be obtained by using a sterile mucus extractor. If cannot be aspirated, instill 4 ml of sterile phosphate buffered saline or 2 drops of PBS into each nostril for nasal aspirate.
- Respiratory viruses can also be recovered from a______ swab preserved in VTM but a______ swab is usually easier to obtain and has been found very satisfactory for the isolation of influenza viruses, measles virus, and respiratory syncytial viruses.
5.______ specimens are required to diagnose respiratory syncytial virus infection, and when necessary to investigate infections caused by influenza and parainfluenza viruses, measles, rubella virus, adenoviruses, and enteroviruses.
Dacron swab; nasopharynx; Calcium alginate
few (5) seconds; VTM
throat; paranasal
Nasopharyngeal
Nasopharyngeal specimens are required to diagnose respiratory_______, and when necessary to investigate infections caused by….
syncytial virus infection
influenza and parainfluenza viruses
measles
rubella virus
adenoviruses
enteroviruses
Nasopharyngeal/Nasal Swab /
Aspirate:
•_______ (superior);_____(convenient)
Bronchoalveolar lavage
• Bronchoscopy (2 viruses)
aspirates; swabs
influenza, adenoviruses
Throat Swab:
• inflamed / purulent areas of____
avoid (4)
OK for (3 viruses)
posterior pharynx
tongue, oral mucosa, teeth &
gums
entero-, adeno- & HSV
Feces
1.Place about____g of feces (small spoon) in a clean, dry, leak-proof container. Deliver to the laboratory as soon as possible.
- If there is likely to be a delay to more than a few hours in the specimens reaching the laboratory, suspend about___g of feces in_________ . If possible centrifuge at 2000 g for 15 minutes, and then transfer the supernatant fluid to a clean leak-proof container.
3.Label and keep at______, if not possible. Send to the virology laboratory in a____.
4.If a feces sample can not be obtained, a_____ swab should be collected and transported in buffered saline.
- The isolation of viruses from the rectal swabs (____cm insertion), however, is less satisfactory than from feces.
6.Fecal specimens are required to diagnose viral_____ especially that caused by rotaviruses (EIA), and to investigate poliomyelitis and other diseases caused by enteroviruses.
4-8 g
one gram; 9 ml of phosphate-buffered saline.
-20 °C or at 4 °C; cold box
rectal; 3-5cm
gastroenteritis
Cerebrospinal Fluid
• Collect_______ml of fluid (or___ml) in a dry, sterile, leak-proof container.
• Refrigerated immediately at____. (Remember in Bacteriology, we don’t refrigerate CSF.)
•Transport in an insulated cold box. UTM not required.
•When meninges are infected, the CSF will contain_____ and the CSF total protein will be raised.
• Frequently isolated: 4 viruses
• Less frequently isolated: 4 viruses
0.5- 1.0 ml; 2-5 ml
4°C
lymphocytes
coxsackievirus (some), echovirus, enterovirus, mumps virus
arboviruses, HSV, LCMV (lymphocytic choriomeningitis virus), rabies
Skin and Ulcer specimens (Rash - maculopapular or vesicular)
1. Gently cleanse area with____
- Maculopapular rash: Disrupt the surface of the lesion firmly at its____ and swab moistened with_____. Place swab in____.
- Vesicular: Sample only fresh vesicles (crusted vesicles may not contain viable virus). Carefully____ vesicle with needle or scalpel blade. Vigorously swab, collect____ at the base of the lesion. For VZV,______ is preferred. Place in UTM.
4.In derma pathology, scraping of an ulcer base to look for _______.
_____, for diagnosis of varicella-zoster virus and herpes.
- Refrigerate immediately at 4°C, and then transport in a cold box.
6.If measles or rubella is suspected, the virus is more likely to be isolated from a______ swab.
- Frequently isolated: enterovirus (some), echovirus, HSV, VZV.
Less frequently: poxviruses
sterile saline.
base; sterile saline; UTM
open; fluid; vesicle aspirate
Tzanck cells, Tzanck smear
paranasal
For VZV,______ aspirate is preferred. Place in UTM.
vesicle
In derma pathology, scraping of an ulcer base to look for________, ______for diagnosis of varicella-zoster virus and herpes.
Tzanck cells - Tzanck smear
If measles or rubella is suspected, the virus is more likely to be isolated from a______ swab.
paranasal
Blood for Serological Tests
1._____ serum specimens are required to diagnose an infection serologically.
2.Collect the sample within____ days of the onset of the symptoms, and the second sample____days later.
- Collect_____ of venous blood in a dry sterile, screw-cap glass tube or bottle.
Two
5; 5-10 days
5-10 ml
Blood
4.A single sample is required for investigating newborn infants with congenital defects, determining immunity to_____ or other viruses.
5.A single serum sample is required when testing for_____ and _____
6.After the blood is collected, collect it in a leak-proof container, refrigerate it at_____ until transport in an ice box to the virology laboratory.
rubella
hepatitis B surface Antigen (HBsAg) and
HIV-1 and HIV-2.
4°C
- a way to identify a virus based upon the cytopathic effects;
also a way to amplify virus if a larger sample is needed for other diagnostic tests.
Tissue culture
Types of tissue culture
Primary
finite
continuous cell lines
- a model mammalian cell line preferred for isolating influenza A and B viruses
MDCK (Madin-Darby canine kidney) cells
- from kidney epithelial cells extracted from an African green monkey, continuous and aneuploid (has an abnormal number of chromosomes, replicated through many cycles of division and not become senescent)
• Vero cells
preferred for
herpes simplex viruses,
poliovirus,
Coxsackie B virus,
RSV,
mumps virus,
rubella virus,
SARS-CoV (BSL2),
lymphocytic choriomeningitis virus (BSL3), among others
Vero cells
examination cells and tissues; blood, lung washings, CSF, cervical swab or scrapings (pap smear), Tzanck smear; observe of CPE
Cytology, Histology
- Cells are grown on slides or coverslips for easy removal from culture
Cell culture and immunofluorescence assays (IFAs) or immunohistochemistry (IC)
Ag & Ab detection
Immuno-serological test methods
detect NA or amplify to detect it.
Rapidly becoming the standards for diagnostic virology. PCR, RT-PCR.
Nucleic Acid Amplification and Detection
can be used to identify morphology of the virus (in cell cultures or directly from tissue specimens)
Electron microscopy
- predict resistant strains (viral gene sequence),
Ex. HIV, hepatitis C and CMV with known resistance mutations
Nucleic Acid Sequencing
IFAs and Indirect IFA (C)
use antibodies conjugated to_____ (______- gives off color when excited by a particular wavelength of light)
•________- best known, blue at 490 nm and green at 519m range.
• FITC-conjugated antibodies specific for a certain virus added in a liquid buffer onto the cells on the slide
Ex. for respiratory syncytial virus, influenza, measles,
mumps, adenovirus
fluorophores
fluorescent dyes
Fluorescein isothiocyanate (FITC)
• performed on cells or tissues (patient specimens or cell cultures infected with patient samples) that have been affixed to slides and exposed to a fixative
IFAs and Indirect IFA (C)
relies upon the same principles as IFA except that the antibody is conjugated to an enzyme (liquid substrate) instead of a fluorescent molecule.
Colored precipitate results are visible using light microscope.
IHC
• Immunoperoxidase with diaminobenzidine (DAB) and hematoxylin as counterstain
[nuclei - blue]
(enzyme substrate - brown)
Chromogenic IHC
• use to detect HBsAg, HSV (Kaposi’s sarcoma), CMV, EBV, Adenovirus, parvovirus B19, yellow fever and Ebola viral antigens, polyomaviruses
IHC
SEROLOGY
• detect viral antigens using antigen-specific antibodies
EIA, ELISA, sandwich ELISA
measures viral antigens
(direct sandwich ELISA) or antiviral antibodies, IgM & IgG (indirect ELISA)
most common screening assay
ELISA
•Blood borne viruses:
HBV, HCV, HIV, HTLV
Measuring the Immune Response to Virus Infection
• establishing the diagnosis of a viral infection is accomplished serologically by demonstrating a rise in antibody titer to the virus or by demonstrating antiviral antibodies of the____ class
•Methods:
neutralization test,
complement fixation test, hemagglutination test, immunofluorescent test,
passive hemagglutination, and immunodiffusion
IgM
• added HIV p24 antigen (earlier identification)
• Before 2014: Western blot was required for diagnosis of HIV, involves binding of patient serum antibodies to HIV proteins separated by protein electrophoresis
4th generation:
•: detect and differentiate antibodies to HIV-1 and HIV-2 and the p24 antigen.
Can yield diagnosis without Western blot. Indeterminate results are investigated using HIV NAAT
5th generation
Western Blot
HIV-1 Western Blot
Lane1:
Lane 2:
Sample A:
Sample B:
Sample C:
(+) Control
(-) Control
(-)
Indeterminate
(+)
