in the name of god diagnostic virology zabih-ollah shoja ph.d. student virology dept. school of...

In The Name Of GODIn The Name Of GOD

Diagnostic VirologyDiagnostic Virology

Zabih-Ollah ShojaZabih-Ollah ShojaPh.D. student Ph.D. student

Virology Dept. School of Public Health, Tehran University of Medical SciencesVirology Dept. School of Public Health, Tehran University of Medical Sciences

History of Diagnostic Virology (1)

The modern era of medical virology began in 1898 with the

discovery by Loeffler and Frosch that foot and mouth disease of cattle.

Viral inclusions had been noted previously in 1892 by

Guarnieri, who described intranuclear and intracytoplasmic inclusions at the base of smallpox lesions.

The earliest methods that were widely used for diagnosing these infections were serologic, directed at the detection of viral antibodies or antigens.


The complement fixation test described by Bedson and Bland in 1929 was first used to detect antibodies to smallpox, vaccinia, and varicella zoster viruses (VZV).

Diagnostic virology leaped forward in 1948 with the report by Weller and Enders of the first growth of pathogenic human viruses in tissue culture, that formed the basis of diagnostic virology for many years.

The first application of fluorescent antibody (FA) staining to diagnostic virology was the detection of influenza virus antigen in nasal smears by Liu (1956).


The development of monoclonal antibodies in the 1970s increased Rapid viral diagnosis during the 1980s as monoclonal antibody-based assays to detect a wide variety of viral antigens directly in clinical specimens.

The introduction of molecular techniques into the diagnostic virology laboratory accelerated with the development of PCR, in 1985, and further with the development of real-time PCR methodology in the late 1990s.

Specimens for Viral Diagnosis (1)

For the diagnosis of acute viral infections, the best specimens are usually obtained from the site of disease;

For example, in the patient with suspected viral meningitis, cerebrospinal fluid (CSF) is the best specimen. In infections involving skin or mucosal surfaces, specimens obtained from those surfaces are usually the only ones required.

Viral titers are highest early in the illness, so that specimens

obtained within the first few days after onset are most likely to be positive.


A specific viral diagnosis depends largely on the quality of the specimen that is received in the laboratory;

Important variables include:

The timing of the specimen in relation to the illness, the type of specimen,The quality and amount of specimen material obtained The time and conditions of transport to the laboratory.


For serologic diagnosis, an acute phase serum specimen should be obtained within the first few days of illness and a convalescent phase specimen 2 to 4 weeks later.

Proper handling of specimens for serologic diagnosis begins with separation of serum or plasma. If testing is performed within several days, the serum or plasma can be stored at 4°C. If a longer delay is involved, the specimen should be frozen at -20°C or -70°C.

For certain viral infections, serologic tests can be performed on saliva or urine.

Methods Used in Diagnostic Virology

Embryonated Egg

Foci formation on the chorioalantoic membrane by vaccinia virus

Small Pox Vaccine Infected Chorioalantoic Membrane

Methods Used in Diagnostic Virology

Viral Culture; Because viruses require cellular machinery for replication,

living systems must be used. Advantages; is an amplification method that increases the amount of the

pathogen, facilitating detection and characterization. provides an isolate of viable virus that and can be stored for

future studies. allow the detection of many different viruses, Even viruses

not previously known can be discovered.

Disadvantages: including requirement for specialized facilities and

expertise, expense, relatively prolonged time to detection, and the relatively limited range of viruses that can be detected.

Viral culture requires more attention to conditions of transport than specimens submitted for detection of viral antigens or nucleic acids, because the viability of the virus must be preserved.

Viral Culture

Types of Cell Culture

Primary cell cultures;

derived directly from the source animal. include primary monkey kidney cells and primary rabbit kidney cells.

Diploid or semicontinuous cells;

are capable of a limited number of passages before undergoing senescence. Include human fibroblast cell cultures such as MRC-5 and WI-38 cells.

Continuous or transformed cell lines;

are immortalized cells that can be passaged without limit. include HEp-2, HeLa, A549, and Madin-Darby canine kidney cells.

Hematopoietic cells; such as peripheral blood mononuclear cells or umbilical cord mononuclear cells, are required for the growth of viruses such as EBV, HHV 6, 8, and HIV.

Specimen Processing and Inoculation

Normally, sterile body fluids (e.g., CSF) can be inoculated directly onto cell cultures.

Urine specimens should have the pH adjusted toward neutrality before inoculation.

from body sites typically contaminated with bacteria (e.g., respiratory or genital specimens) are treated with antibiotics before inoculation to prevent bacterial or fungal overgrowth.

After inoculation, cultures are incubated at 35°C to 37°C and inspected periodically (e.g., once daily or every other day).

Respiratory cultures directed at detection of influenza or rhinoviruses can be incubated at 33°C.

Detection of Viral Replication

Cytopathic Effect:

The growth of viruses in cell culture is often associated with morphologic changes in the infected cells (CPE). The features of the CPE may allow recognition of the infecting virus (Syncytium).

Important characteristics:

a. cell culture types are affected.

b. the timing and rate of progression.

c. the distribution (focal, diffuse, limited to

the margins of the cell culture).

a. the nature of the morphologic changes.

Syncytium and CPE

Hemadsorption

Paramyxoviruses, orthomyxoviruses, may produce only minimal CPE and, instead, are routinely detected by testing the cultures for their ability to bind red blood cells.

This occurs because certain viruses express on the surface of infected cells viral proteins that bind to erythrocyte membranes. The binding is specific for the erythrocytes of certain species, such as guinea pig, rat, or monkey.

Interference Antigen or Nucleic Acid Detection Electron microscopy



Shell Vial Culture:

A modified cell culture in which the specimen is centrifuged onto the cell monolayer and viral growth is detected by antigen detection procedures.

Originally for CMV, but has subsequently been applied to many other viruses, including herpes simplex virus (HSV), VZV, respiratory viruses, and Enteroviruses.

The main advantage: it can decrease the time required to detect the presence of the virus.

Shell Vial Culture


Mixed Cell Culture:

Which involve simultaneous growth of more than one cell culture type in a single shell vial, have been developed to facilitate the isolation in the same culture of multiple viruses that require different cells for growth.

For example, a commercial system called R-Mix combines human adenocarcinoma (A549) and mink lung cell lines (mv1Lu) for growth of respiratory viruses.

After incubation, the R-Mix cells are stained with a mixture of monoclonal antibodies specific for seven respiratory viruses.

Genetically Engineered Cell Lines:

Modify cell lines either to make them susceptible to viruses to which they are not otherwise susceptible or to create novel means of detecting viral growth.

consists of baby hamster kidney (BHK) cells that have been transfected with the b2-galactosidase gene from Escherichia coli under the control of a promoter from the herpes simplex gene UL39.


Genetically Engineered Cell Lines

Electron Microscopy

for the direct visualization of viral particles in specimen.

Advantages include speed, lack of requirement for viral viability, and that many different kinds of viral particles can potentially be seen (nature of the particle).

Disadvantages include the cost and complexity of maintaining an electron microscopy, the need for a skilled operator, and limited sensitivity related to the relatively high concentration of viral particles.

1. Direct Specimen Examination (EM)

diagnostic virology is the evaluation of stool specimens from patients with suspected viral gastroenteritis.

rotaviruses, enteric strains of adenovirus, noroviruses, and astroviruses, are not readily cultivable.

All be seen by negatively stained using phosphotungstic acid or uranyl acetate.

Rotaviruses and adenoviruses are easily seen. Noroviruses is enhanced by preincubation with immune

serum (immune electron microscopy).

2. Examination of Fixed Tissue (EM)

3. Examination of Infected Cell Cultures(EM)

when they cannot be identified by other methods.

Although identification to the species level is not possible, identification of a virus family based on morphology can provide a starting point for more detailed identification.

light Microscopy The most characteristic signs Of viral infection are inclusion

bodies (composed of masses of virions), multinucleated cells,

and syncytial cells. (nucleus or the cytoplasm).

VIRUS Nuclear Cytoplasmic

Herpes simplex *

Varicella zoster *

Cytomegalovirus *

Adenovirus *

Polyomaviruses (JC and BK) *

Parvovirus B19 *

Poxviruses *

Measles * *

Parainfluenza *

Rabies *

Cytology

Tzanck Smear: An example of cytologic staining that used for the rapid detection

of HSV or VZV infection in skin lesions. The presence of multinucleated giant cells or intranuclear

inclusions suggests the presence of HSV or VZV. The main advantages of the technique are that it is rapid and does

not require sophisticated equipment.

Papanicolaou Staining:

Human papillomavirus infection produces characteristic changes in keratinocytes with a koilocytosis

Urine Cytology:

urinary sediment may reveal intranuclear inclusions indicative of infection with either CMV or the polyomaviruses, JC and BK.

Cytology

Histology

provide unique information about the role of viral infection in producing tissue inflammation and injury, and can be very useful in distinguishing between asymptomatic viral shedding and clinically significant infection.

is particularly useful for CMV infections in which prolonged viral shedding can occur without producing disease. For example, the presence of cytomegalic inclusion cells in tissue obtained by biopsy or at autopsy (gold standard).

Antigen Detection

Antigen detection methods can provide diagnostic information within a few hours of the receipt of the specimen in the laboratory.

The lack of requirement for virus viability is important advantage compared with viral culture, especially when specimen transport time is prolonged.

Methods used for viral antigen detection include

FA staining, immunoperoxidase staining, and enzyme immunoassay, …

Nucleic Acid Amplification Assays

The earliest attempts at nucleic acid-based diagnosis involved direct hybridization of nucleic acid probes to viral nucleic acids present in clinical specimens.

Techniques that detect specific nucleic acid sequences by amplification improved of dignosis.

Nucleic acid amplification assays are attractive for viruses that are difficult or impossible to cultivate, viruses that grow very slowly in culture, and viruses for which antigen detection cannot be applied because of antigenic diversity or because the level of viral antigen in clinical specimens is too low to permit successful detection.

Target Amplification Assays

PCR

RT-PCR has been applied to the detection of numerous RNA viruses,

including HIV, hepatitis C virus (HCV), enteroviruses, RSV, influenza and parainfluenza viruses, rotavirus, and other RNA viruses that cause gastroenteritis.

RT-PCR can also be applied to the detection of viral messenger RNA. diagnosis of infection caused by viruses that have a latent phase in their life cycle. detection of viral DNA might not distinguish between latent and productive infection, whereas detection of a messenger RNA (mRNA) expressed only in productive infection would be evidence of active viral infection.

nucleic acid sequence based amplification( Transcription-mediated assay)

It is an isothermal nucleic acid amplification method that amplifies RNA in a manner analogous to the replication of retroviruses

The NASBA reaction mixture contains oligonucleotide primers and three enzymes: RT, RNase H and T7 RNA polymerase for target-specific amplification

This process occurs at 41ºC Detection of NASBA products has been reported using probe-

hybridization and electrochemiluminescence More recently, detection ‘real-time’ using molecular beacons

has been described

Signal Amplification AssaysBranched chain DNA assay (detection of HIV

and hepatitis C RNA).

Signal Amplification AssaysHybrid capture assay (detection of the DNA of

HPV, CMV, and HBV).

Release and denature acid nucleic

Hybridize RNA Probe with target DNA

Capture RNA: DNA hybrids onto a solid phase

React captured hybridized With Multiple Ab conjugates

Detect amplified chemiluminescent signal

Multiplex PCR

In multiplex PCR more than one target sequence can be amplified by including more than one pair of primers in the reaction.

Choose Loci Position Primers Design Primers Develop PCR Conditions Separately

Nested PCR PCR is performed on the primary PCR product using a new set

of internal primers. For increased sensitivity and …

Real-time PCR

advantages The chance for contamination

decreased, cause the systems are closed.

Rapid cycling times (1 hour). Of great importance,

quantitation of PCR targets. Very sensitive

Non Specific:

DNA-binding agents

(SYBR Green )

Specific:

Hydrolysis probes

(TaqMan, Beacons,

Scorpions,Sunrise)

Hybridization probe

(Light Cycler or Adjacent)

SYBR Green

TaqMan 5’ exonuclease assay

Molecular Beacons

Scorpion primers

Hybridization probe (Adjacent)

Southern Hybridization

requires that 10,000 or more copies of the target sequence (marker) be present in order for the test to be positive (limitation in test's sensitivity)

can NOT detect point mutation It is a simple, reliable and sensitive method with an

incredibly broad applications including general DNA analysis, gene cloning, screening and isolation, DNA mapping, mutant detection, identification of genetic and infectious diseases.

use a gel to separate DNA fragmentsdenature DNA after separationtransfer DNA strands to membraneattach DNA strands to membranehybridize and detect bound probe

Southern Hybridization

Transferring DNA from Gel to membrane

gel(from side) separation by size

transfer DNAfrom gel

nitrocellulose or nylonmembrane(from side)

Prehybridization and hybridization

Use a probe to detect the target DNA Block unoccupied sites with innocuous DNA Incubate blot with labeled probe

• Appropriate temperature

• Appropriate [salt]

Wash off unbound probe Detect location of probe

USES

Identify mutations, deletions, and gene rearrangements

Used in prognosis of cancer and in prenatal diagnosis of genetic diseases Leukemias

Diagnosis of HIV-1 and infectious disease

In situ hybridization (ISH)

ISH can identify sites of gene expression, tissue localization of mRNA and identification & localization of viral infection

There are 2 methods of ISH : RISH, using radioactive and FISH using fluorescent probes

Steps Part A. Tissue Fixation, Embedding, Sectioning and

Mounting of Sections on Slides Preparation of Coated Glass Slides Fixation of Cultured Cells on Slides Tissue Fixation, Embedding, Sectioning and Mounting of

Sections on Slides

Part B. In Situ Hybridization and Detection Using Isotopic Probes

Dewaxing of Sections Protease Digestion DNase Treatment for in Situ Hybridization of RNA RNase Treatment for in Situ Hybridization of DNA Preparation of Probes In Situ Hybridization Detection of Hybridized Signals

In situ hybridization and detection of signal

1. Prehybridization treatment with heat2. Hybridization buffer containing probe is used while

heating slides to denature probe and target molecule3. Removing unhybridizaed RNA by RNAse treatment4. Dehydrate slides and proceed to emulsion

autoradiography 5. Expose the X-ray film to the slides at 4C for 1-10 days

In situ PCR or RT-PCR hybridization

In this technique virus infection can be detected and localized rapidly

Low copy DNA or RNA were first amplified in the cell, then detected by ISH

Trapping amplified molecules in cellular compartment needs appropriate fixative

formalin can extensively polymerize proteins and cross link NAs and provide a barrier for amplified DNAs

Performing PCR in situ

1. Prepare a PCR cocktail2. Add it to slides and cover it by cover slip and seal

the edges3. Carry out 25 cycles of PCR amplification

Tips: a hot start PCR (pre-heating)at 70ºC prevent mispairing between primer and template

4. Final step is detection of product by RISH or FISH

Microarray Technology

A high throughput technology that allows detection of thousands of genes simultaneously

Multiple samples at the same time Differing expression of genes between tissues and

disease states identification of gene copies in a genome mutation and polymorphism detection sequencing, diagnostic tools for diseases, and drug discovery

Similar to NorthernBase-Pairing, hybridization between nucleic acids

Major differences from NorthernDetects thousands of genes simultaneously/individualProbes fixation on glass slide / nylon membraneTarget samples labeling with fluorescent/radioactive

dNTP

Principle

What are microarrays

A microarray is typically a glass slide on to which DNA molecules are fixed at specific locations called spots

contain thousands of spots and each spot may contain a few million copies of identical DNA molecules (probe) that uniquely correspond to a gene.

The DNA in a spot: may either be genomic DNA (Probe cDNA 500~5,000 bases long) short stretch of oligo-nucleotide strands (20~80-mer oligos) The spots are printed on to the glass slide by a robot or are

synthesized by the process of photolithography.

How do they work

most popular applications:

is to compare expression of a set of genes in cells maintained in two particular condition

DNA microarray due to its unrestricted capacity to accommodate hundreds to thousands of individual gene probes

Allows the simultaneous detection any amplifiable pathogen present in a specimen.

This technology is ideal for the extensive identification and differentiation of various pathogens and their strains.

The technique is rapidly evolving from a novel research technology to a practical tool for the identification and the detection of viruses and the genotyping of influenza viruses and human immunodeficiency virus (HIV) type 1.

DNA-microarrays in virology

DNA-microarrays in virology

Wang et al. (2002) have described a microarray spotted with 70-mer oligonucleotides which represent the five most conserved sequences of each virus of interest.

The Chip contains 1600 different probes. Following PCR amplification in the clinical specimen using random primers, hybridisation onto the microarray was able to identify respiratory viruses in the enterovirus, rhinovirus, paramyxovirus, adenovirus and herpesvirus families.

Mapping of Genomic Segments of Influenza B Virus Strains by an Oligonucleotide Microarray Method

DNA Microarrays for Virus Detection in Cases of Central Nervous System Infection (38 gene)

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Serology

the first methods used for the specific diagnosis of viral infections.

Serologic diagnosis is important for viruses that cannot be cultured readily or for which culture is slow or otherwise impractical.

Kinetics of the Antibody Response (1)

Virus-specific IgM antibodies are often detectable for a short period before virus-specific IgG antibodies.

The IgM response tends to decline within approximately 1 to 2 months, although low levels can persist for 1 year or more in some viral infections.

IgG antibodies are much more long-lasting, and can persist for the life span of the individual.

The absence of virus-specific IgG and IgM antibodies signifies susceptibility to infection;

Kinetics of the Antibody Response(2)

The presence of virus-specific IgM antibodies, with or without virus-specific IgG antibodies, signifies current or very recent infection; and the presence of virus-specific IgG, but not IgM antibodies, signifies past infection.

An alternative serologic approach to the diagnosis of recent infection is based on the concept that the avidity of antigen-specific antibodies increases as the immune response matures.

Thus, antibodies produced early after infection have low affinity for the causative agent, and antibodies produced late after infection have high affinity. Detection of low-affinity antibodies has been used to diagnosis recent infection with CMV, rubella and HIV.

Serology in Reinfection and Reactivation

In both of these cases, the infection occurs in an individual with preexisting, virus-specific IgG antibodies.

In reinfection an response may occur that results in an increase in the level of virus-specific IgG antibodies with continued absence of virus-specific IgM antibodies.

Reactivation of latent infection, for example in the case of some herpesvirus infections, may result in the appearance of virus-specific IgM as well as IgG antibodies.

Serology in Chronic Infections

The interpretation of serology is unique for viruses that typically cause chronic infection (e.g., HIV) and (HTLV)-1 and -2.

For these agents, the detection of antibodies of any isotype to these viruses (in the absence of artificial immunization) virtually always signifies current infection.

In the case of HCV, the confirmed presence of antibodies to the virus corresponds to active infection in approximately 85% of individuals.

Serologic Assays (1)

Binding Assays:

directly measure binding of antibodies to viral antigens. Include EIA, radioimmunoassay (RIA), and the indirect immunofluorescent antibody assay (IFA).

Immunobinding Assays:

The western blot


Functional Assays: detection of specific activities resulting binding Ab to viral Ag. neutralization assay, which measures the ability of antibodies

to block viral infectivity. complement fixation assay hemagglutination-inhibition assay

Agglutination Assays: Viral antigens can be bound to a variety of particles, including

fixed erythrocytes and latex particles. they are simplicity, rapid and do not require sophisticated equipment.

hemagglutination-inhibition assay

in which the presence of antiviral antibodies is detected

by their ability to block virus-induced hemagglutination

Antigen + Antibody + Complemen

Sheep RBC + Antibody to Sheep RBC

complement fixation assay


Saliva and Urine Assays:Virus-specific antibodies can often be detected in

saliva and urine.

These body substances are attractive for use in serologic assays because they avoid the need for phlebotomy.

Assays for HIV antibodies in these fluids are commercially available and have performance characteristics similar to assays carried out on serum


Cerebrospinal Fluid Serology: Serologic testing can be applied to CSF for the diagnosis of

central nervous system (CNS) infection. For unusual viruses (e.g., rabies), the presence of any virus-

specific antibodies within the CSF is diagnostic of active infection.

For the diagnosis of encephalitis caused by the alphaviruses, bunyaviruses, or flaviviruses, the presence of virus-specific antibodies in CSF is highly suspicious and the presence of virus-specific IgM antibodies in CSF is diagnostic.

Cerebrospinal Fluid Serology

common viruses (e.g., herpesviruses) or respiratory viruses, the mere presence of virus-specific antibodies in CSF is not diagnostic of CNS infection because antibodies produced in the blood are present in the CSF even in the absence of CNS infection.

Intrathecal synthesis of a specific antiviral antibody is evaluated by determining the quotient of two ratios:

a. the ratio of specific antiviral antibody level in CSF to the level in serum,

b. the ratio of total IgG in CSF to total IgG in serum

A quotient greater than 1.5 is evidence of intrathecal antibody synthesis of the specific antibody.

References 1. Fields virology; 2007

2. Flint virology; 20043. Steven J Read, David Burnett and Colin G Fink. Molecular techniques

for clinical diagnostic virology J. Clin. Pathol. 2000;53;502-506

4. James J. Hughes. Application of quality systems to virology testing . Journal of Clinical Virology 31 (2004) 92–95

5. Guy Vernet. Molecular diagnostics in virology. Journal of Clinical Virology xxx (2004) xxx–xxx

6. Elizabeth Valentine-Thon. Quality control in nucleic acid testing*/where do we stand. Journal of Clinical Virology 25 (2002) S13 - S21

7. Hubert G.M. Niesters. Clinical virology in real time. Journal of Clinical Virology 25 (2002) S3/S12

8. Mikael Kubista and et al. The real-time polymerase chain reaction. Molecular Aspects of Medicine 27 (2006) 95–125

9. Christian M. Leutenegger. The Real-Time TaqMan PCR and Applications. Veterinary Sciences Tomorrow – Issue 1 – January 2001

10. ROBERT S. LANCIOTTI AND AMY J. KERST. Nucleic Acid Sequence-Based Amplification Assays for Rapid Detection of West Nile and St. Louis Encephalitis Viruses. JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 2001, p. 4506–4513

11. M C Edwards and R A Gibbs. Multiplex PCR: advantages, development, and applications. PCR Methods Appl. 1994 3: 65-75

12. ELFATH M. ELNIFRO AND ET AL. Multiplex PCR: Optimization and Application in Diagnostic Virology. CLINICAL MICROBIOLOGY REVIEWS, Oct. 2000, p. 559–570

13. David Wang and et al. Microarray-based detection and genotyping of viral pathogens. PNAS November 26, 2002: 99(24); 15687–15692

14. In Situ Hybridization Protocols (Methods in Molecular Biology) (Hardcover)

15. Wu, William, et al. 2004, Gene Biotechnology, second edition, CRC press LLC.

16. Yury S. Boriskin and et al. DNA Microarrays for Virus Detection in Cases of Central Nervous System Infection. JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 2004, p. 5811–5818

17. Wang D, Coscoy L, Zylberberg M, Avila PC, Boushey HA, Ganem D, et al. Microarray-based detection and genotyping of viral pathogens. Proc Natl Acad Sci USA 2002;99:15687–92.

18. Clewley JP. A role for arrays in clinical virology: fact or fiction. J Clin Virol 2004;29:2–12.

19. Anna V. Ivshina and et al. Mapping of Genomic Segments of Influenza B Virus Strains by an Oligonucleotide Microarray Method. JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 2004, p. 5793–5801

Thanks for your attentionThanks for your attention

Herpes Simplex Virus and Varicella Zoster Virus

Mucocutaneous Infections: direct detection by culture, viral antigen, or viral nucleic acid. Specimens are obtained by unroofing a vesicle and using a

swab to soak up vesicular fluid as well as to scrape the base of the lesion.

Calcium alginate swabs should not be used because this material inhibits the growth of HSV .

HSV grows very well in cell culture, producing CPE in 1 to 3 days.

A variety of cell culture types can be used, but rabbit kidney, mink lung, and guinea pig embryo cells are particularly susceptible.

shell vial culture techniques, or the ELVIS genetically engineered cell line. Mixed cultures containing CV-1 and MRC-5 cells with the capability to detect either HSV or VZV. FA staining of the infected cell culture.

The sensitivity of cell culture techniques for HSV is strongly influenced by the stage of the lesion, with higher sensitivity for vesicular and pustular lesions than for lesions that are crusted.

Varicella zoster virus is more difficult than HSV to grow in culture.

This virus grows only in human fibroblast cells and monkey kidney cells, and typically requires 4 to 8 days to become detectable. The sensitivity of culture detection is lower than for HSV



Rapid Diagnosis: by FA staining.

Nucleic Acid Detection: real-time PCR ; advantages of speed, high sensitivity, and

ability to distinguish between HSV-1 and 2. In one study, real-time PCR was 28% more sensitive than

culture. Some real-time assays for VZV have the ability to distinguish

between wild-type virus and the vaccine strain


Serology: has little role in the diagnosis of acute HSV infection because

viral culture or antigen detection is usually successful; because

acute and convalescent phase specimens, difficulties in interpretation related to both cross-reactions and asymptomatic reactivations.

VZV serology is occasionally useful for diagnosis of acute varicella if appropriate specimens were not obtained for culture and antigen detection or if those tests were negative despite a clinical impression that varicella was the true diagnosis.

B Virus

B virus infection; bite or other exposure to infectious secretions or excretions from a monkey infected with B virus.

specimens should be obtained using a Dacron or cotton swab from Mucocutaneous lesions.

CSF and conjunctival and pharyngeal swabs can also sometimes yield the virus, which grows in several types of primary and continuous monkey kidney cell cultures.

Biosafety level 3 is required for laboratories culturing B virus from clinical samples.

PCR can also be used to detect B virus. Acute and convalescent serum specimens should also be

obtained, although cross-reactions with HSV can make interpretation problematic.

Cytomegalovirus Culture: culture is the traditional gold standard for the diagnosis of

CMV. antigen or nucleic acid detection are faster, more sensitive,

and easier to quantitate. CMV grows only in human fibroblast cells, and typically

requires 7 to 21 days to exhibit cytopathic effect. shell vial technique is a replacement for conventional culture because it provides results within 24 to 48 hours.

The interpretation of CMV cultures can be problematic because of frequent shedding of the virus in saliva and urine in asymptomatic individuals.

Cultures of blood are more helpful diagnostically, because positive blood cultures are virtually never found in asymptomatic, nonimmunocompromised individuals.

Cytomegalovirus

pp65 Antigenemia Assay: In this assay, leukocytes are separated from other blood

elements, spotted onto a microscope slide, and stained using a monoclonal antibody to pp65, the CMV lower matrix phosphoprotein.

availability of results within a few hours of receipt of the blood sample in the laboratory, sensitivity exceeding that of culture, capability for providing quantitative data, and a strong, although not perfect correlation, with the presence of clinically significant infection.

Nucleic Acid Detection Assays

Serology: Cytomegalovirus serology is of less value for the diagnosis of

infections in immunocompromised patients than direct tests for the virus.

is useful, however, for the diagnosis of CMV infection in the normal host and for defining CMV immune status, for example in blood donors or for transplant donors and recipients.

EIA is more sensitive than the complement fixation test, which was widely used in the past.

CMV avidity assays

Cytomegalovirus

Cytomegalovirus in the Normal Host: a mononucleosis-like illness. the heterophile antibody test is

negative. It should be noted that acute EBV infection can also yield a

false positive finding in the CMV IgM test. In patients with CMV mononucleosis, CMV is regularly

detected in the urine by culture, and often in the blood by culture, the pp65 antigenemia, or PCR

Cytomegalovirus

Cytomegalovirus in the Immunocompromised Host: important cause of opportunistic infection in patients who

have received either solid organ or hematopoietic stem cell transplants and AIDS.

quantitative test performed on blood. PCR is the most sensitive test.

The hybrid capture assay and the pp65 antigenemia assay become positive several days to 1 week after PCR and occasionally fail to detect CMV antigenemia before the onset of symptoms.

Culture is the least sensitive, and often the last test to become positive.

Cytomegalovirus

The diagnosis of localized CMV infection:

areas such as the respiratory or gastrointestinal is best made by histologic examination of tissue from the site of disease.

Diagnosis of CNS infection is best achieved by performing PCR on CSF.

The most severe form of CMV encephalitis, ventriculoencephalitis, is strongly associated with very high levels of CMV DNA in CSF

Cytomegalovirus

Congenital Infection: diagnosed by culture or PCR performed on urine or saliva

within the first 2 weeks of life.

Quantitative testing of blood can be useful because symptomatic disease is associated with higher levels, and elevated levels may predict subsequent hearing loss.

In utero CMV infection can be diagnosed by culture or PCR performed on amniotic fluid.

Cytomegalovirus

Human Herpes Virus-6 and 7

both viruses are universal in the population. virus may continue to be present for an extended period

following initial infection. asymptomatic reactivation may occur. Culture is impractical for most clinical laboratories because it

requires the use of umbilical cord lymphocytes, which are not readily available to most such laboratories.

Assays for HHV-6 antibodies are most useful in young children who are undergoing primary infection. In these children, seroconversion is diagnostic of acute infection.

Interpretation of serologic findings in children younger than 6 months of age can be complicated by the presence of maternal antibodies.

Type of Infection

Whole Blood PCR

Plasma PCR Saliva PCR HHV-6 IgG

Primary, acute stage

Positive, high level

Positive Negative Negative

Primary, convalescent stage

Positive Negative Positive Positive

Reactivation Positive Positive or Negative

Positive Positive

Human Herpes Virus-6 and 7

Epstein-Barr Virus

The cultivation of EBV is carried out by inoculating specimen into a suspension culture of umbilical cord lymphocytes.

Evidence for the presence of EBV growing in the culture is the immortalization of the cells and the demonstration of the presence of EBV antigens in the immortalized cells (practical).

infectious mononucleosis are diagnosed with presence of atypical lymphocytes in peripheral blood plus heterophile antibodies.

Paul-Bunnell-Davidson test.

Epstein-Barr Virus

Specific Serology: IgM anti-VCA is useful in defining acute infection. because IgM

anti-VCA is usually detectable with the onset of clinical manifestations, and remains detectable for only several months.

IgG anti-VCA is useful in defining EBV immune status, because these antibodies are detectable for the life of the individual.

Anti-EA arise within a few weeks of infection, but are not detected in all patients with acute infectious mononucleosis.

Anti-NA arise later after infection than anti-EA, and persist for life.

Immunocompromised Patients: In patients with AIDS, EBV is associated with primary CNS

lymphoma PCR to detect EBV DNA in CSF is useful for making this diagnosis.

In transplant recipients, EBV is associated with posttransplant lymphoproliferative disorder (PTLD). The diagnosis of PTLD is confirmed by demonstrating EBV antigens or nucleic acid in a biopsy specimen of lymphoid tissue showing the appropriate histologic findings.

quantitative testing can identify a subgroup of patients with high levels who either have PTLD or are at high risk of developing it

Epstein-Barr Virus

Respiratory Viral Infections

Culture: The influenza and parainfluenza viruses replicate best in

primary monkey kidney cells, although continuous cell lines (e.g., Madin-Darby canine kidney) can be used for influenza and LLC-MK2 for parainfluenza.

Serum-free medium and some strains of influenza virus replicate best at 33Â°C.

without producing CPE and hemadsorption with guinea pig erythrocytes is used for detection (24 hours).

RSV replicates optimally in HEp-2 cells. very labile. delayed transport result in failure to isolate RSV.

Adenoviruses replicate optimally in human embryonic kidney cells.

Rhinoviruses replicate only in human diploid fibroblast cells. Incubation at 33°C to 34°C.

human metapneumovirus grown in monkey kidney cells (e.g., LLC-MK2), but growth is slow and clearly less sensitive than detection by molecular methods.

SARS replicates in Vero and other monkey kidney cells, including fetal rhesus monkey kidney cells.

Recently, the process of culturing respiratory viruses has been simplified by the development of mixed cell culture systems


Antigen Detection: be used to achieve rapid diagnosis of RSV, influenza,

parainfluenza, and adenovirus respiratory tract infection. Viral antigens are visualized by FA staining of respiratory

epithelial cells.

nasopharyngeal aspirate, wash, or swab, Tracheal aspirate and bronchoalveolar lavage specimens. Aspirates and washes provide a higher cellular yield and have higher viral titers than swabs.

Rhinoviruses and coronaviruses are not detected by antigen detection methods because of antigenic diversity and lack of availability of appropriate antibodies.


Nucleic Acid Amplification: The advantages of these assays are increased sensitivity

compared with conventional methods and the ability through multiplex methods to detect multiple viruses in the same sample.

nucleic acid amplification can detect viruses such as HMPV, coronaviruses, and human bocavirus that are difficult or impossible to detect by other methods.

A advantage is that nucleic acid amplification facilitates the detection of respiratory viruses from nonrespiratory sites.


Gastrointestinal Viral Infections

Culture: rotaviruses, enteric adenoviruses (especially, serotypes 40

and 41), noroviruses, sapoviruses, astroviruses, and possibly toroviruses that is not readily cultivable.

Electron Microscopy: Negative staining with uranyl acetate or phosphotungstic acid

can be performed directly on stool specimens. especially the noroviruses and sapoviruses, are better

visualized using immune electron microscopy

Antigen Detection: Many commercial assays that use for rotavirus detection, and

at least one commercial adenovirus assay is available. Antigen detection assays for astrovirus have been used in

investigations

Nucleic Acid Amplification: In rotaviruses antigen detection assays better than RT-PCR

because of the adequate sensitivity and greater simplicity. RT-PCR has been shown to be more sensitive than EIA for

detection of astroviruses. RT-PCR is the method of choice for detection of noroviruses.

Gastrointestinal Viral Infections

Viral Meningitis

Enteroviral Meningitis: echoviruses and coxsackieviruses are the most common. PCR assays that amplify a segment of the 5' nontranslated

region of the enterovirus genome. viral culture of CSF, which has a sensitivity of 70% or less, is

not successful for some Coxsackie A strains, and requires at least several days to detect viral growth.

Herpes Simplex Virus Meningitis: Most HSV meningitis is associated with HSV-2. PCR appears to be both sensitive and specific for HSV

meningitis.

Varicella Zoster Virus Meningitis: as a complication of either primary or reactivation infection.

PCR can be used to amplify VZV DNA from the CSF.

The results of VZV PCR performed on CSF must be interpreted with the knowledge that VZV DNA is present in some cases of zoster without neurologic complications.

PCR may be uniquely useful in recognition of cases of VZV meningitis without cutaneous or skin lesions.

Viral Meningitis

Viral Encephalitis

Herpes Simplex Virus Encephalitis:

PCR performed on CSF has revolutionized, which previously required a brain biopsy for laboratory diagnosis.

In a study meta-analysis sensitivity and specificity of HSV PCR for the diagnosis of HSV encephalitis were found to be 96% and 99%, respectively.

Arbovirus Encephalitis: Diagnosis of arboviral encephalitis is best accomplished by

serologic testing. EIA or IFA assays used to detect arboviral-specific IgM

antibodies, which are diagnostic of recent infection when detected in either CSF or serum.

use of nucleic acid amplification techniques to detect West Nile virus RNA can be useful in making the diagnosis in immunocompromised patients in whom the serologic response may be delayed or absent.

Viral Encephalitis

Rabies encephalitis: The diagnosis of rabies is accomplished by a variety of

methods, depending on the stage of the infection. earliest phase detection by culture, antigen detection using FA

staining, or nucleic acid amplification. saliva and CSF used for culture and RT-PCR and skin (usually

from the nape of the neck) for FA staining. brain biopsy should be analyzed for rabies antigen by FA

staining. After the eighth day of illness, rabies antibodies are diagnostic if

the patient had not received rabies immunization. (Ab in CSF)

Viral Encephalitis

Other Causes of Meningitis or Encephalitis in Immunocompetent Hosts

Include EBV, HHV-6, B virus, lymphocytic choriomeningitis virus (LCMV), measles, mumps, and rubella viruses, influenza virus, adenoviruses, and Nipah virus.

be diagnosed by a combination of culture, serology, and nucleic acid amplification.

Opportunistic Viral Infections of the CNS

Cytomegalovirus meningoencephalitis is best diagnosed by using quantitative PCR on CSF.

EBV is strongly associated with primary CNS lymphoma in patients with AIDS that PCR performed on CSF can detect EBV DNA in most cases.

PCR for JC virus is useful for diagnosing progressive multifocal leukoencephalopathy.

Viral Hepatitis

Hepatitis A

is diagnosed using assays for hepatitis A IgM antibodies.

and remains positive for several months.

Assays for total antibodies to hepatitis A virus are used to define previous exposure and immunity.

Acute Hepatitis B: is diagnosed in patients with jaundice or fatigue and high ALT

levels. Evidence of acute HBV infection is obtained by the detection

of HBsAg and anti-HBc antibody IgM. Coinfection with HDV is evaluated at this stage by assaying

for serum HDAg, anti-HDV IgM, and HDV RNA.

Viral Hepatitis

Chronic Hepatitis B: persistence of serum HBsAg for >6 months(asymptomatic). The diagnosis is usually made by the presence of serum HBsAg

and elevation in ALT levels. a close monitoring of HBV DNA and ALT levels. In chronic HBV carriers who present with an acute exacerbation of

transaminase levels and other symptoms of acute hepatitis It is necessary to rule out superinfection by HAV, HCV, or HDV, especially in patients such as IV drug users, patients requiring transfusions, and so on.

In addition, ALT elevation may be a consequence of the immune attack on infected cells preceding anti-HBe seroconversion and remission of liver disease. In this case, the ALT flare is accompanied by a decrease in serum viral load.

The other possibility is reactivation of viral replication. It occurs in inactive carriers with low levels HBV DNA in whom the rise in viral load precedes the elevation in ALT levels.

Viral Hepatitis

Hepatitis C;Serologic Assays: detection anti-HCV in plasma or serum include both EIA and

chemiluminescence immunoassays(NS3, NS4, and NS5). confirmatory serologic test for anti-HCV is the strip immunoblot

assay (RIBA HCV recombinant antigens and synthetic peptides ). Nucleic Acid Detection: RT-PCR, real-time PCR , transcription-mediated amplification and

and signal amplification methods. HCV genome sequences are analyzed for genotyping by direct

sequencing or reverse hybridization or by means of a competitive EIA detecting genotype-specific antibodies.

Viral Hepatitis

Hepatitis D and E: is diagnosed by the detection of antiviral antibodies. HDV RNA can be detected by RT-PCR, and may be useful for

defining active infection.

Hepatitis E is diagnosed using an EIA that measures antibodies to hepatitis E virus.

The presence of anti-HEV IgM antibodies indicates recent infection.

Hepatitis E RNA can be detected in blood during the acute phase of the illness.

Viral Hepatitis

Human Papillomaviruses

Human papillomaviruses cannot be grown in cell culture.

findings on cervical Pap smears referred to as koilocytosis are indicative of HPV infection.

PCR (gene encoding the L1 major capsid protein) and the hybrid capture assay have been used to detect HPV DNA in patient specimens.

The hybrid capture assay is less sensitive than PCR but more sensitive than a southern blot.

Human Polyomaviruses

BK virus grows in human embryonic kidney cells, and a shell vial culture method.

JC virus grows only in human fetal brain cultures and a human fetal cell line called SVG.

PCR assay for JC virus is performed on CSF from patients suspected of having progressive multifocal leukoencephalopathy (PML).

BK virus PCR is performed on urine and/or plasma. (one third of all recipients of kidney transplants).

Quantitative testing may be helpful, because the level in the urine predicts the appearance in plasma, and the level in plasma may predict the occurrence of BK nephropathy.

Measles

grown in a variety of human and simian cells. Specimens include nasopharyngeal and conjunctival secretions,

blood, urine, and CSF. Rapid diagnosis can be accomplished by FA staining of

nasopharyngeal secretions or urine. rapid diagnosis is for measles-specific IgM antibodies, are

always detectable within several days of the appearance of rash. The diagnosis can also be made by comparing measles

antibody titers in acute and convalescent phase specimens. The presence of measles virus IgG antibodies in serum is

evidence of measles-specific immunity. SSPE is diagnosed by high levels of measles Ab in CSF. by using RT-PCR

Rubella

African green monkey kidney, Vero, or RK-13 cells. specimens: nasopharyngeal or throat secretions, urine, stool,

blood, and joint fluid. virus can grow without producing CPE, and must be detected

by using the interference assay that confirmed by specific FA staining or nucleic acid detection techniques.

rubella virus-specific IgM antibodies, which are usually detectable within a few days of onset of the rash.

Rubella virus antibody titers can also be compared in acute and convalescent specimens

IgG avidity

Mumps

Rhesus monkey kidney cells and human embryonic kidney cells.

Specimens: saliva, urine, and CSF. The virus grows slowly, and often requires hemadsorption with

guinea pig erythrocytes for detection. shell vial assay. virus-specific IgM antibodies assay. Mumps virus antibody titers can also be compared in acute

and convalescent specimens. Cross-reactivity with parainfluenza viruses. RT-PCR can also be used for sensitive detection of mumps

RNA in oropharyngeal secretions, urine, or CSF.

Parvovirus B19

The virus cannot presently be cultured in routine diagnostic virology. parvovirus B19-specific IgM antibodies, which are present within

several days of the onset of symptoms. may cross-react with other viruses, especially rubella, measles, EBV, and CMV.

comparing parvovirus B19-specific antibody titers in acute and convalescent specimens.

In Hemolytic crisis used nucleic acid amplification. Because patients with fifth disease are usually viremic, the

diagnosis can be established by using PCR. chronic hypoplastic anemia in immunocompromised patients, PCR

is the best approach for making a virologic diagnosis. IgG for immunity.

Enteroviruses

RT-PCR or NASBA to detect enteroviral RNA in CSF. Enteroviral infections of body sites other than the CNS can

be diagnosed by culture or by nucleic acid amplification. stool and nasopharyngeal or throat secretions.

Coxsackie B viruses grow well in primary monkey or human embryonic kidney cells and continuous monkey and human cell lines.

Coxsackie A viruses require newborn mouse inoculation for growth.

echoviruses grow in human fibroblast cells, primary monkey or human embryonic kidney cells.

A549 cells combined with BGM kidney cells that engineered to express decay accelerating factor(receptor enterovirus) and increases the susceptibility of the cells to enterovirus infection.

Viruses growing in culture can by FA staining, but type-specific identification requires the laborious neutralization test.

be cultured from respiratory tract specimens for 1 to 2 weeks after infection, and for 4 to 6 weeks from stool specimens.

Enteroviruses

Congenital and Neonatal Infection

acquisition of fetal samples: chorionic villus sampling amniocentesis to obtain amniotic

fluid, and cordocentesis to sample fetal blood. Chorionic villus sampling provides a fetal sample from the first

trimester of pregnancy, whereas amniocentesis and cordocentesis are usually performed during the second or third trimesters.

diagnosis in utero or neonatal infection: viral culture, viral nucleic acid amplification assays, and tests

for virus-specific IgM antibodies.

Virus In utero Newborn

CMV Culture or PCR on amniotic fluid

Culture of urine during first 2 weeks of life

VZV PCR on amniotic fluid Fluorescent antibody (FA) stain or PCR or culture of lesion

HSV No experience #95. Slide 95

Parvovirus B19

PCR on amniotic fluid Parvovirus B19 IgM or PCR on serum

HBV Not applicable HBsAg on serum

HIV Contraindicated PCR or RT-PCR

Rubella Rubella-specific IgM on fetal blood, culture or RT-PCR on amniotic fluid

Rubella-specific IgM, persistence of rubella-specific IgG, culture of urine, pharynx, conjunctiva, blood, stool, or CSF

Enteroviruses Not applicable Culture or RT-PCR of stool, nasopharyngeal secretions, blood, or urine

HCV No information RT-PCR performed at 6-18 months of age. Serum EIA for anti-HCV performed after 12 months of age

LCMV No information LCMV-specific IgG and IgM

Typical neonatal infection (usually acquired intra- or postpartum) is diagnosed by culture or FA stain or culture of lesion.

Cultures of mouth, nasopharynx, conjunctiva, skin, urine, cerebrospinal fluid (CSF), and blood may also be useful.

PCR performed on CSF may help in detection of involvement of the central nervous system.

PCR performed on blood may help in recognition of disseminated infection, especially in cases lacking cutaneous lesions.

Rare cases of HSV acquired in utero may be recognized by detection of HSV-specific IgM antibodies detected in blood sample obtained during first week of life.

HIV

Antibody Assays: is performed using EIA. detect all serotypes of HIV-1, with the

exception of some group O viruses. Current HIV EIA typically become positive approximately 22

days after infection. western blot testing is required to confirm positive EIA results. western blot if positive, has extremely high sensitivity and

specificity. The major difficulty with this approach is that occasional western blots are indeterminate. Resolved by molecular tests (see below) or by repeating the EIA and western blot after several months.

rapid or simple tests results within a short time (e.g., <1 hour). HIV is considered confirmed if the specimen is positive using two different rapid or simple tests.

agglutination assays and rapid tests. The U. S. Public Health Service has recommended the use of such tests in selected situations for testing individuals who may not return to learn of test results.

HIV antibody testing can be performed on oral transudate or urine for screening in situations in which it is not practical to draw blood.

HIV

p24 Antigen Assays: Assays for HIV p24 antigen in serum can detect HIV infection

approximately 6 days earlier than the antibody EIA. Plasma HIV viral load assays have replaced most of the use

of this assay.

HIV

HIV Nucleic Acid Amplification Assays: The HIV DNA PCR assays that detect proviral DNA are used to

detect infection in infants born to HIV-infected mothers. estimates are that HIV DNA PCR is positive in approximately

40% of cases within the first few days of life and greater than 90% by 2 weeks of age.

Assays for plasma HIV RNA; viral load and to evaluate the response to treatment (RT-PCR, branched chain DNA, and transcription-mediated amplification).

These assays can detect as few as 40 copies of HIV RNA per milliliter of plasma. (the earliest detection of HIV after infection).

they are now used for screening of blood donor units as well as for the diagnosis of individuals with the acute retroviral syndrome.

Application Test

Routine diagnosis Antibody enzyme immunoassay (EIA)

Confirmation of positive EIA Western blot

Resolution of indeterminate western blot DNA polymerase chain reaction (PCR)

Blood donor screening Antibody EIA, p24 EIA, viral load assay

Infant born to infected mother DNA PCR

Acute retroviral syndrome p24 EIA or viral load assay*

Prognosis Viral load assay

Response to therapy Viral load assay

Resistance to antiretroviral drugs Genotypic or phenotypic susceptibility test

Exotic Viral Infections

Virus or Syndrome Location TestsSin Nombre virus (hantavirus

pulmonary syndrome)Western United States Virus-specific IgM, RT-PCR

Other hantaviruses Asia, Europe Virus-specific IgM

Colorado tick fever Western United States Culture of blood clot, FA stain of erythrocytes, RT-PCR

Dengue fever Asia, Central and South America

Virus-specific IgM, RT-PCR

Hemorrhagic fevers (filoviruses [Ebola and Marburg],

arenaviruses [Lassa and South American hemorrhagic fevers], yellow fever, Congo-Crimean

hemorrhagic fever)

Africa, South America, Asia

Virus-specific IgM, culture of serum, RT-PCR, EM of urine or

blood (Ebola and Marburg), antigen detection assays

Rift Valley fever Africa Virus-specific IgM, culture, RT-PCR

Alphaviruses (Chikungunya, Oâ€™nyong nyong, sindbis,

Mayaro, Ross River

Africa, Europe, Asia, Central and South America, Australia

Virus-specific IgM, culture, RT-PCR

in the name of god diagnostic virology zabih-ollah shoja ph.d. student virology dept. school of...

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