Viral InfectionsViral Infections

Terry Kotrla, MS, MT(ASCP)BBTerry Kotrla, MS, MT(ASCP)BB

Herpes Virus GroupHerpes Virus Group

Produce a variety of diseases.Produce a variety of diseases. May result in sub-clinical infectionsMay result in sub-clinical infections May be reactivated under May be reactivated under

appropriate conditions.appropriate conditions.

Herpes Virus GroupHerpes Virus Group

We will discuss the following:We will discuss the following:– Epstein-Barr virus– Cytomegalovirus– Herpes simplex virus type I and II– Varicella-zoster virus

Epstein-Barr Virus (EBV)Epstein-Barr Virus (EBV)

Spread through oral transmission Cause of Infectious

Mononucleosis. Other Diseases include:

– African or Burkitt’s lymphoma– Nasopharyngeal carcinoma– B cell lymphoma

Epstein-Barr virus

African or Burkitt’s LymphomaAfrican or Burkitt’s Lymphoma– malignant B-cell neoplasm malignant B-cell neoplasm – presents as a rapidly growing tumour of presents as a rapidly growing tumour of

the jaw, face or eyethe jaw, face or eye– grows very quickly, and without grows very quickly, and without

treatment most children die within a few treatment most children die within a few months months

– Epstein-Barr virus (EBV) has been Epstein-Barr virus (EBV) has been strongly implicated strongly implicated

African or Burkitt’s LymphomaAfrican or Burkitt’s Lymphoma

Although BL is a very rapidly growing tumour Although BL is a very rapidly growing tumour it responds well to treatment. it responds well to treatment.

Three pictures: before treatment, 3 days and Three pictures: before treatment, 3 days and 6 days after treatment6 days after treatment

Nasopharyngeal CarcinomaNasopharyngeal Carcinoma Endemic in South China, Africa, Arctic Endemic in South China, Africa, Arctic

EskimosEskimos This is a malignant tumour of the This is a malignant tumour of the

squamous epithelium of the nasopharynx.squamous epithelium of the nasopharynx. 100% contain EBV DNA100% contain EBV DNA Rates are less than 1 per 100,000 in most Rates are less than 1 per 100,000 in most

populations populations Nasopharyngeal carcinomas are found in Nasopharyngeal carcinomas are found in

association with reactivation of latent association with reactivation of latent Epstein-Barr Virus.  Epstein-Barr Virus. 

The exact mechanisms of association are The exact mechanisms of association are unknown unknown

B-Cell LymphomaB-Cell Lymphoma

In most individuals infected with In most individuals infected with EBVEBV, the , the virus is present in the virus is present in the BB-cells, which are -cells, which are normally controlled by T-lymphocytes normally controlled by T-lymphocytes

When T-When T-cellcell deficiency exists, one clone of deficiency exists, one clone of EBVEBV-infected -infected BB-lymphocytes escapes -lymphocytes escapes immune surveillance to become immune surveillance to become autonomously proliferating. autonomously proliferating.

EBVEBV induced induced B cellB cell lymphomas are most lymphomas are most prevalent in immunocompromised prevalent in immunocompromised patients. patients.

Oral Hairy Cell LeukoplakiaOral Hairy Cell Leukoplakia

Viral infection of the oral cavity. Viral infection of the oral cavity. Indicator of HIV infection as well as of a Indicator of HIV infection as well as of a

person's lessening or weakening immunityperson's lessening or weakening immunity

Infectious MononucleosisInfectious Mononucleosis

4 to 7 week incubation Acute self-limiting infection of the RE system Enlarged lymph nodes in the neck. Sore throat, fever, rash Malaise, lethargy, extreme tiredness Liver and spleen involvement and

enlargement Hematology: High WBC, over 20% atypical

reactive lymphocytes also known as Downey cells.

Infectious MononucleosisInfectious Mononucleosis

Downey cells may be presentDowney cells may be present

Heterophile Heterophile Antigens/AntibodiesAntigens/Antibodies

Heterophile antigens are a group of similar antigens found in unrelated animals, IE, man, sheep, horse, dog cat, mouse.

Heterophile antibodies produced against heterophile antigens of one species will cross react with others.

Heterophile Heterophile Antigens/AntibodiesAntigens/Antibodies

Forssman antigen is an example of a heterophile antigen and is found on the RBCs of many species (guinea pig, dog, cat, mouse, sheep, fowl, horse)

Forssman antibodies formed against Forssman antigens will agglutinate sheep RBCs.

Paul Bunnell TestPaul Bunnell Test The original Paul-Bunnell test was a simple titration of The original Paul-Bunnell test was a simple titration of

sheep cell agglutinins but this procedure was subsequently sheep cell agglutinins but this procedure was subsequently modified in order to distinguish between sheep cell modified in order to distinguish between sheep cell agglutinins formed in IM and the Forssman-type antibodies agglutinins formed in IM and the Forssman-type antibodies found in normal serum, serum sickness and in certain other found in normal serum, serum sickness and in certain other conditions.conditions.

Tissues rich in Forssman antigen (guinea pig kidney) absorb Tissues rich in Forssman antigen (guinea pig kidney) absorb Forssman antibodies but do not affect the heterophil Forssman antibodies but do not affect the heterophil antibodies in IM.antibodies in IM.

Heterophil antibodies are absorbed by beef cells,Heterophil antibodies are absorbed by beef cells, Forssman hapten is a glycolipid usually associated with a Forssman hapten is a glycolipid usually associated with a

protein, the determinant being largely carbohydrate and protein, the determinant being largely carbohydrate and therefore heat stable.therefore heat stable.

Davidsohn DifferentialDavidsohn Differential The principle behind the Paul-Bunnell-Davidsohn test is that The principle behind the Paul-Bunnell-Davidsohn test is that

the two types of sheep agglutinins are distinguished by the two types of sheep agglutinins are distinguished by titrating them before and after absorption with guinea pig titrating them before and after absorption with guinea pig kidney and ox cells.kidney and ox cells.

Patients serum containing antibodies due to IM is added to Patients serum containing antibodies due to IM is added to guinea pig kidney cells. These antibodies are not absorbed guinea pig kidney cells. These antibodies are not absorbed by the kidney cells. These antibodies then react with Beef by the kidney cells. These antibodies then react with Beef (Ox) red blood cells which causes agglutination and is a (Ox) red blood cells which causes agglutination and is a positive test for IM.positive test for IM.

Patients serum containing Forssman antibodies are added Patients serum containing Forssman antibodies are added to guinea pig kidney cells. Antibodies are absorbed by the to guinea pig kidney cells. Antibodies are absorbed by the kidney cells. These antibodies are then allowed to react kidney cells. These antibodies are then allowed to react with Beef red blood cells which does not cause with Beef red blood cells which does not cause agglutination. This is a positive test for Forssman antigens.agglutination. This is a positive test for Forssman antigens.

Davidsohn DifferentialDavidsohn Differential** To be considered absorbed there must be greater than a three tube To be considered absorbed there must be greater than a three tube

difference between the presumptive titer and the differential titer.difference between the presumptive titer and the differential titer.

Heterophil Antibody

------------------------ Infectious Mono

Kidney Extract ------------------ Not Absorbed

Beef Erythrocyte --------------------- Absorbed

Forssman

Absorbed

Not Absorbed

Serum Sickness

Absorbed

Absorbed

Davidsohn DifferentialDavidsohn Differential

AdvantagesAdvantages

When properly performed, When properly performed, this test is specific for this test is specific for Infectious Mononucleosis Infectious Mononucleosis and false-positive results and false-positive results are rare.are rare.

DisadvantagesDisadvantages

Davidsohn Differential test Davidsohn Differential test is very time consuming is very time consuming and burdensome.and burdensome.

Infectious Mono Slide TestsInfectious Mono Slide Tests

It was discovered that horse RBCs possess antigens which react with the antibody associated with IM.

Patient serum mixed with horse RBCs, agglutination is positive.

Latex agglutination Not diagnostic, must look at total

clinical picture.

EBV Specific AntibodiesEBV Specific Antibodies

EBV specific antibodies may be measured. Pattern of appearance of EBV antigens. Most valuable is IgM antibody to viral capsid

antigen (VCA), indicates a current infection (best marker), lasts about 12 weeks.

Can also detect anti-early antigen (EA) (recent infection) and anti EB nuclear antigen (EBNA) (older infection).

ELISA and IFA most commonly used

Cytomegalovirus

Transmission occurs from person to person.

Close intimate contactClose intimate contact– Sexual contactSexual contact– PernatallyPernatally– Breast milkBreast milk– Organ transplantOrgan transplant– Blood transfusionBlood transfusion

CMV CMV Clinical course

Symptoms resemble IM In babies may cause life threatening

illness Patients with deficient immune

systems AIDS patients Transplant patientsTransplant patients

CMV CMV Immunologic response

Test for CMV antibody using paired serum samples

IgM antibodies produced against early and intermediate-early (IE) CMV antigens, last for 3 to 4 months.

IgG appear shortly after and peak at 2 to 3 months.

CMV CMV Laboratory Diagnosis

Range from culture and cytologic techniques to DNA probes, PCR and serologic techniques.

Detection of antibodies indicator of recent or active infection.

Viral cultures

Microscopic examination of biopsy specimens

CMV Lab DiagnosisCMV Lab Diagnosis

Detection of CMV antigen in cells using IFAusing IFA

ELISA to detect antibody to CMVELISA to detect antibody to CMV OtherOther

– fluorescence assays, – indirect hemagglutination, and– latex agglutination

False positives can occur due to RA and Epstein-Barr antibodies

Herpes Simplex Virus (HSV)

Most exposed in childhoodMost exposed in childhood Possesses viral latency – hibernationPossesses viral latency – hibernation Two types: HSV-1 and HSV-2

HSV-1

Transmitted from person to person by saliva or direct contact.

Cold sores around the mouth most common.

Reactivation - may have several episodes of cold sores during a lifetime

HSV-1HSV-1

SymptomsSymptoms– tingling – NumbnessNumbness– ItchingItching

Blister forms, breaks, crusts overBlister forms, breaks, crusts over Reactivation usually caused by stress.Reactivation usually caused by stress. Conjunctivitis, keratitis and herpetic

whitlow may occur.

HSV-2HSV-2 Results in Herpes genitalis - lesions BELOW the Results in Herpes genitalis - lesions BELOW the

waist.waist. Transmitted Transmitted intimate sexual contact or

perinatally. SymptomsSymptoms

– PainPain– TendernessTenderness– ItchItch– FeverFever– HeadacheHeadache– LymphadenopathyLymphadenopathy– MalaiseMalaise

HSV-2HSV-2

Blisters appearBlisters appear– Males – penisMales – penis– Females – vagina and cervixFemales – vagina and cervix– Both – thighs buttocksBoth – thighs buttocks

Painful, lasts 1-3 weeksPainful, lasts 1-3 weeks Virus lies dormant in nearby nerves Virus lies dormant in nearby nerves

and reactivated.and reactivated.

HSV – 2HSV – 2

Can be fatal in infantsCan be fatal in infants Woman with active infection needs Woman with active infection needs

C-section.C-section. Infants with localized infections have Infants with localized infections have

70% mortality rate70% mortality rate Disseminated neonatal herpes most Disseminated neonatal herpes most

lethal form.lethal form.

Neonatal HerpesNeonatal Herpes

Laboratory TestingLaboratory Testing

Recovery of virus from cultureRecovery of virus from culture Direct examination of cells from lesion Direct examination of cells from lesion

using IF or immunoperoxidase stainusing IF or immunoperoxidase stain DNA probesDNA probes ELISAELISA Latex agglutinationLatex agglutination RIARIA Indirect IFIndirect IF Serology NOT very usefulSerology NOT very useful

Varicella-Zoster Virus

Two different manifestations of the same virus.– Varicella is the primary infection,

causes chicken pox– Herpes Zoster causes shingles and is

due to reactivation of the latent virus

VaricellaVaricella

Fever and vesicular exanthemaFever and vesicular exanthema Small, itchy blisters surrounded by Small, itchy blisters surrounded by

inflamed skin.inflamed skin. Begins as one or two lesions and Begins as one or two lesions and

spreads.spreads. Number of lesions vary greatly.Number of lesions vary greatly. Blister dries out and forms a scab.Blister dries out and forms a scab.

Chicken PoxChicken Pox

Chicken PoxChicken Pox

Secondary complications due to infection most common.– May also result in pneumonia,

encephalitis and hepatitis.– Very serious for immunocompromised

children Vaccine now available

ShinglesShingles

Chicken pox – virus goes latentChicken pox – virus goes latent Reactivated later in lifeReactivated later in life

– Weakened immune systemWeakened immune system– AgingAging– Other factorsOther factors

ShinglesShingles

The typical rash of shingles begins as redness(erythema) followed by the appearance of blisters..

Eruptions follow the path of an infected nerve.

The trunk is the area affected in 50% to 60% of cases.

Skin may be extremely sensitive to touch

ShinglesShingles

ShinglesShingles

ShinglesShingles

Laboratory TestingLaboratory Testing

Important to distinguish VZV from other infections– PCR – Direct Fluorescent Antibody staining Direct Fluorescent Antibody staining – Viral cultureViral culture– IgG and IgM antibody test by ELISAIgG and IgM antibody test by ELISA

Rubella Virus

RNA virus with 3 major structural proteins, E1, E2, and C.

Incubation 2- 3 weeks Highly contagious, spread through

respiratory tract. Causes German measles Rubella vaccine has resulted in 99%

decline in infections.

RubellaRubella

Congenital rubella Congenital Rubella Syndrom most serious.Congenital Rubella Syndrom most serious. Fetus infected during first trimester.Fetus infected during first trimester. result in miscarriage or stillbirth, live-born serious birth defects or dying. 20% of the children born after such an

infection suffer the severe congenital abnormalities

10-20% of these children die within the first year of life.

Rubella vaccine contraindicated during pregnancy.

Rubella SyndromeRubella Syndrome

Lab testingLab testing

IgG and IgM antibodies may form at same time

IgM antibodies persist for 4 to 5 weeks, IgG for life.

Performed primarily for diagnosis of acquired infections and to determine immune status of pregnant patients.

Some tests detect IgG antibodies, other IgM.

Laboratory Testing - RubellaLaboratory Testing - Rubella

Methods include: – hemagglutination inhibition, – passive hemagglutination, – neutralization, – hemolysis in gel, – complement fixation, – fluorescence immunoassay, – RIA, – ELISA and – latex agglutination.

Rubeola

Single stranded RNA virus best known for its typical skin rash

Primarily respiratory infection Incubation approximately 10 days,

ranges from 8-13. Rash appears at about day 14. Airborne precautions

RubeolaRubeola

Symptoms include – irritability, – runny nose, – eyes that are red and sensitive to light,– hacking cough, and – high fever

RubeolaRubeola

Fever peaks with the appearance of the rash.

Rash typically begins on the forehead, then spreads downward over the face, neck, and body.

Rash appears on face first and consists of large flat red to brown blotches that often flow into one another

Rash fades in the same order that it appeared

RubeolaRubeola

Complications

Croup bronchitis bronchiolitis pneumonia conjunctivitis myocarditis Hepatitis encephalitis

RubeolaRubeola

More susceptible to ear infections or pneumonias

Disease can be severe, with bronchopneumonia or brain inflammation

May lead to death in approximately 2 of every 1,000 cases.

Most severe in adultsMost severe in adults

Measles vaccine

Live attenuatedLive attenuated DO NOT give to:DO NOT give to:

– pregnant women, – persons with active tuberculosis, – leukemia, – lymphoma, – depressed immune systems. – People with egg allergies

Measles vaccineMeasles vaccine

Occasionally causes side effects in persons with no underlying health problems,

In about 10% of cases there is a fever between 5 and 12 days after vaccination,

In about 5% of cases there is a rash.

Laboratory Testing

Serology testing provides best means of confirming a measles diagnosis

Methods to detect rubeola antibodies include: – hemagglutination inhibition, – endpoint neutralization, – complement fixation, – IFA and – ELISA.

Laboratory TestingLaboratory Testing

Diagnosis confirmed by presence of Rubeola specific IgM antibodies antibodies

or four-fold rise in IgG antibody titer in paired samples taken after rash to 10 to 30 days later.

IgM test highly depended on time of sample collection with 3-11 days after rash being optimal.

IgM false positive due to RA.

MumpsMumps

Single stranded RNA virus. Mumps is transmitted by direct

contact with saliva and discharges from the nose and throat

iIncubation 16-18 days. Virus can infect many parts of the

body, especially the parotid salivary glands.glands.

MumpsMumps

Glands usually become increasingly swollen and painful over a period of 1 to 3 days

Pain gets worse Both the left and right parotid glands

may be affected

MumpsMumps

MumpsMumps

Mumps - Mumps - Complications

inflammation and swelling of the brain Mumps in adolescent and adult males

may also result in the development of orchitis

May affect the pancreas or, in females, the ovaries

Infection in pregnant women may result in increased risk for fetal death

Laboratory TestingLaboratory Testing

complement fixation hemagglutination inhibition hemolysis-in-gel neutralization assys IFA and ELISA

Laboratory TestingLaboratory Testing Current or recent infections indicated by presence

of specific IgM antibody in single sample which can be detected within 5 days of illness.

Fourfold rise in specific IgG antibody in 2 samples collected during acute and convalescent phases

Fluorescent antibody staining for mumps antigens Cross-reactivity between antibodies to mumps

and parainfluenza viruses has been reported in tests for IgG, not a problem since symptoms differ.