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					                   In The Name Of GOD




     Diagnostic Virology


                             Zabih-Ollah Shoja
                                Ph.D. student
Virology 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.
      History of Diagnostic Virology (2)
 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).
      History of Diagnostic Virology (3)

 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.
      Specimens for Viral Diagnosis (2)

 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.
      Specimens for Viral Diagnosis (3)
 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.
                  Viral Culture
 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.
                   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
         Detection of Viral Replication
 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
         Detection of Viral Replication

 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
         Detection of Viral Replication

 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.
        Detection of Viral Replication

 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.
                      Cytology

 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.
                     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 Assays
Branched chain DNA assay (detection of HIV
          and hepatitis C RNA).
      Signal Amplification Assays
Hybrid capture assay (detection of the DNA of
           HPV, CMV, and HBV).
      Release and                                Detect amplified
      denature                                   chemiluminescent
      acid nucleic                               signal




            Hybridize RNA
            Probe with                       React captured hybridized
            target DNA                       With Multiple Ab conjugates




                            Capture RNA: DNA hybrids
                            onto a solid phase
                    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                         Non Specific:
 The chance for contamination      DNA-binding agents
  decreased, cause the systems         (SYBR Green )
  are closed.
 Rapid cycling times (1 hour).        Specific:
 Of great importance,
                                      Hydrolysis probes
  quantitation of PCR targets.
 Very sensitive                     (TaqMan, Beacons,
                                     Scorpions,Sunrise)
                                     Hybridization probe
                                  (Light Cycler or Adjacent)
SYBR Green
TaqMan 5’ exonuclease assay
Molecular Beacons
Scorpion primers
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.
        Southern Hybridization



use a gel to separate DNA fragments
denature DNA after separation
transfer DNA strands to membrane
attach DNA strands to membrane
hybridize and detect bound probe
Transferring DNA from Gel to membrane

                                    nitrocellulose
                                     or nylon
                                    membrane
                                    (from side)


                                     transfer
                                       DNA
                                     from gel
     gel
 (from side)   separation by size
 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 heat
2. Hybridization buffer containing probe is used while
   heating slides to denature probe and target molecule
3. Removing unhybridizaed RNA by RNAse treatment
4. 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 cocktail
2. Add it to slides and cover it by cover slip and seal
   the edges
3. 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
                     Principle

 Similar to Northern
Base-Pairing, hybridization between nucleic acids

 Major differences from Northern
Detects thousands of genes simultaneously/individual
Probes fixation on glass slide / nylon membrane
Target samples labeling with fluorescent/radioactive
  dNTP
                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-microarrays in virology
 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
 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)
Subgrid (VZV)   Metagrid (VZV)
3
      ×
    × × ×
      ×
            5

3
      ×
    × × ×
      ×
            5

3
      ×
    × × ×
      ×
            5
Subgrid (HSV-2)   Metagrid (HSV-2)

3
      ×
    × × ×
      ×
            5

3
      ×
    × × ×
      ×
            5
                      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
                 Serologic Assays (2)
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
complement fixation assay

    Antigen + Antibody   Sheep RBC + Antibody
    + Complemen          to Sheep RBC
              Serologic Assays (4)
 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
                 Serologic Assays (5)

 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; 2004
3.   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
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    MICROBIOLOGY, Dec. 2001, p. 4506–4513
11. M C Edwards and R A Gibbs. Multiplex PCR: advantages,
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14. In Situ Hybridization Protocols (Methods in Molecular Biology)
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15. Wu, William, et al. 2004, Gene Biotechnology, second edition, CRC
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    CLINICAL MICROBIOLOGY, Dec. 2004, p. 5811–5818
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    5801
Thanks 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.
Herpes Simplex Virus and Varicella Zoster Virus

 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
Herpes Simplex Virus and Varicella Zoster Virus

 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
  Herpes Simplex Virus and Varicella Zoster Virus

 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
                   Cytomegalovirus
 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.
          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.
              Human Herpes Virus-6 and 7
Type of        Whole     Plasma PCR       Saliva PCR   HHV-6 IgG
Infection      Blood PCR

Primary,       Positive,    Positive      Negative     Negative
acute stage    high level

Primary,     Positive       Negative      Positive     Positive
convalescent
stage
Reactivation   Positive     Positive or   Positive     Positive
                            Negative
                  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.
                  Epstein-Barr Virus
 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
            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 33°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.
           Respiratory Viral Infections
 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
            Respiratory Viral Infections
 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.
            Respiratory Viral Infections
 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
        Gastrointestinal Viral Infections
 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.
                     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.
                     Viral 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 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.
                   Viral Encephalitis
 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)
  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.
                     Viral Hepatitis
 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.
             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.
                        Enteroviruses
 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.
       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      Culture of urine during first 2 weeks of
                fluid                           life
VZV             PCR on amniotic fluid           Fluorescent antibody (FA) stain or PCR or
                                                culture of lesion
HSV             No experience                   #95. Slide 95
Parvovirus      PCR on amniotic fluid           Parvovirus B19 IgM or PCR on serum
B19
HBV             Not applicable                  HBsAg on serum
HIV             Contraindicated                 PCR or RT-PCR
Rubella         Rubella-specific IgM on fetal   Rubella-specific IgM, persistence of
                blood, culture or RT-PCR on     rubella-specific IgG, culture of urine,
                amniotic fluid                  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.
                               HIV
 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
 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                     Tests
 Sin Nombre virus (hantavirus    Western United States    Virus-specific IgM, RT-PCR
     pulmonary syndrome)
      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     Virus-specific IgM, RT-PCR
                                    South America
Hemorrhagic fevers (filoviruses Africa, South America, Virus-specific IgM, culture of
     [Ebola and Marburg],                 Asia         serum, RT-PCR, EM of urine or
arenaviruses [Lassa and South                           blood (Ebola and Marburg),
American hemorrhagic fevers],                             antigen detection assays
 yellow fever, Congo-Crimean
      hemorrhagic fever)
        Rift Valley fever                Africa        Virus-specific IgM, culture, RT-
                                                                     PCR
  Alphaviruses (Chikungunya,     Africa, Europe, Asia, Virus-specific IgM, culture, RT-
  O’nyong nyong, sindbis,       Central and South                  PCR
      Mayaro, Ross River          America, Australia

				
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