this is about chicken pox lab daignostic method and prevention.

1. CHICKEN POX
(VARICELLA)
LAB DIAGNOSIS AND
PREVENTION
Presented by
Dr. Samarita Banik
MD Resident , Phase-A, Year -1
Department of Microbiology And Immuno
BSMMU

2. INTRODUCTION
• Chicken Pox , also known as Varicella, is an acute, highly contagious disease caused
by Varicella Zoster Virus.
• It is characterized by generalized vesicular eruption of the skin and mucous
membrane.
• It is distributed worldwide and is a common epidemic disease of childhood( most
cases occur in children <10 years)
• The disease may be Severe in adult and immunocompromised individuals.
• Usually occurs in spring and winter season .

4. CHICKEN POX :ORIGIN OF THE TERM
• One is that it’s from the blisters that are seen
with the illness. These are red spots which are
5mm to 10mm wide ,were once thought to
look like chickpeas
• Another theory is that the rash of chicken pox
looks like the peck marks caused by a chicken.

5. VIRUS PROPERTIES
• Belong to the alpha herpes virus sub family
of herpes viruses
• Double stranded DNA enveloped virus
• Genome size 125 kbp,
• Single serotype only, although there is some
cross reaction with HSV

6. TRANSMISSION AND PATHOGENESIS
• Chicken pox is very contagious and spreads from person to person by sneezing ,
coughing , contaminated clothing and direct contact with open blisters.
Day 0-3: Infection of conjunctiva and mucosa of URT. then viral replication in regional lymph nodes
occur
Day 4-6:primary viremia spreads virus and leads to replication in liver and spleen
Day10-12:secondary viremia
Day 14:infected mononuclear cells transports virus to the skin and typical rash develops.

7. CLINICAL MANIFESTATIONS
• After an average incubation period of 14 to 16
days (range: 10–21 days), clinically apparent
disease ensues, with characteristic skin lesions in
varying stages of development and resolution.
Cutaneous lesions begin as macules and rapidly
progress to papules, vesicles, pustules, and scabs.
Fever and rash last approximately 5 days, with
rash being more concentrated on the trunk and
head than on the extremities. With wild-type
disease, most children develop 250 to 500 skin
lesions, and lesions frequently develop in the
mouth, conjunctivae, or other mucosal sites.
Bullous or hemorrhagic lesions occur rarely.

10. LAB DIAGNOSIS
• Chicken pox is usually diagnosed clinically. Lab diagnosis usually not required.
• Lab testing is recommended to:
• Confirm suspected cases of varicella
• Confirm varicella as the cause of outbreaks
• Confirm varicella in severe cases or unusual cases
• Determine susceptibility to varicella
• Determine if suspected vaccine related adverse events caused by vaccine strain VZV

11. LAB DIAGNOSIS CONT..
• For rapid diagnosis following tests can be done-
• 1) direct virus demonstration by
• -Microscopy of Tzanck’s smear
• -Detection of viral antigen by DIF
• -Detection of nucleic acid by PCR(Gold standard)
• 2)virus isolation in tissue culture
• 3)serology-Detection of VZV antibodies

12. SPECIMENS
Vesicular fluid
scabs
Blood

13. COLLECTION INSTRUCTIONS
Collecting crusts(scabs):
Crusts can be ideal specimen and can be lifted off the skin and placed in between two glass slides in a
sturdy cardboard container or in a break resistant , snap cap or screw top tube and ship dry.
Polyester swab method –vesicular lesions
Use a sterile needle to unroof the top of vesicle. Vigorously swab the base of the lesion, applying
enough pressure to collect epithelial cells without causing bleeding and collect vesicular fluid . Place
the swab into labelled tube. Ship dry. Do not place transport media into tube.
Glass slide method-maculopapular lesions(if no vesicles are present)
Rake edge of sterile slide over the maculopapular lesion, enough so skin cells are gathered on the slide.
Then use a single swab to scrub the lesion and also to gather the material on the edge of the slide. Ship
dry.

15. • Blood collection – collect whole venous blood in
serum separator vacutainer tube. Allow the
specimen to clot by standing at room temperature
for at least 30 mins. After the clot has been formed,
tube can be centrifuged at approximately 1100-
1300Xg for 10 mins. The clot will have passed to the
bottom of the tube, leaving the serum above.
• CSF collection-CSF specimens should be collected
under sterile conditions and placed in a leak proof
container.
• Minimum volume required -0.5 -1ml(serum, CSF)
• Transport media –not required

16. DEMONSTRATION OF VZV DNA BY
PCR
• Demonstration of VZV DNA by PCR tests from a clinical specimen, ideally scabs,
vesicular fluid or cells from the base of a lesion is the preferred method for varicella
diagnosis. PCR is also used to detect viral genome in CSF , BAL, tissues, EDTA blood
or amniotic fluids etc. PCR is also useful for confirming breakthrough varicella. It is
the most rapid and sensitive method for VZV detection.it can also be used to
distinguish rashes due to wild type VZV from rashes vaccine type strain VZV.

17. MICROSCOPY
Preparation of Tzanck’s smear-
1. Unroof vesicle and scrape base with sterile number 15 scalpel blade.
2. Smear onto a clean glass slide
3. Fix with gentle heat or air dry
4. Fix with methanol
5. Stain with Giemsa, methylene blue or Wright’s stain
6. Microscopic examination using oil immersion lens.
Findings: Characteristic appearance of cells( Multinucleated giant cells) in specimens of skin
lesions. Multinucleated giant cell contain acidophilic intranuclear body(Cowdry type A inclusions)
may be found in necrotic tissue.

19. DIRECT FLUORESCENT ANTIBODY
ASSAY
• Direct fluorescent antibody assays are used
to diagnose varicella zoster virus in skin
lesion.
• Principle: A monoclonal antibody directed
against a unique antigen on the VZV is
conjugated with a fluorescent marker that
can be seen with a fluorescence
microscope. Bright apple green
fluorescence is observed.

20. VIRUS ISOLATION BY CELL CULTURE
• Isolation of VZV through culture of vesicular fluid provides a definitive diagnosis, however,
culturing for VZV is technically difficult and cultures are positive 40% of the time.
The “gold standard” for diagnosis of VZV infections was once virus isolation. This requires the
use of fresh vesicular fluid, which is inoculated immediately onto monolayers of susceptible
cells, such as human embryonic lung fibroblasts (HELF), and incubated at 35 to 37°C for 3 to
7 days; cultures are then examined microscopically for cytopathic effect. This technique is
costly and slow. Worse, because VZV is a labile virus, cultures can fail to reveal infection
even when VZV is present. The slow growth of VZV in cell culture is also a serious handicap
for patient care, which often involves decisions that have to be made quickly. Virus isolation is
thus not much used for diagnosis today, although it is still useful in the determination of
whether a putative VZV infection is resistant to antiviral drugs.

22. DETECTION OF VZV ANTIBODIES
• Detection of antibodies can done by Enzyme Linked Immunosorbent Assay
(ELISA), fluorescent antibody to membrane antigen (FAMA) assay etc methods.
• Detection of IgM can provide evidence for recent active VZV infection, but cannot
discriminate between a primary infection and re infection or reactivation from
latency since specific IgM antibodies are transiently produced on each exposure to
VZV. IgM tests are also inherently prone to poor specificity.
• Paired acute and convalescent sera showing a four fold rise in IgG antibodies have
excellent specificity for varicella , but are not as sensitive as PCR. People with prior
history of vaccination or disease history may have very high baseline titers, and may
not achieve a four fold increase in the covalescent sera.

25. RECOMBINASE-AIDED
AMPLIFICATION-LATERAL FLOW
• A rapid detection method for VZV using recombinase-aided amplification-lateral
flow (RAA-LF) technology has been developed. The gene of interest is the VZV open
reading frame 63 (ORF63), which encodes an immediate early protein (IE63) that is
synthesized during lytic infection. IE63 is also expressed during the latent phase,
indicating a critical role in the maintenance and establishment of virus latency. The
whole process, from the RAA reaction to the visualization of the band on the LF
dipstick, can be completed in less than 30 min.

27. • In the era of universal varicella vaccination, diagnosis of varicella is challenging,
especially for breakthrough cases. We sought to clarify the reliability of direct
varicella-zoster virus (VZV) loop-mediated isothermal amplification (LAMP) and
DermaQuick® VZV using the immunochromatography technique as rapid
diagnostic tests for varicella. In addition, the usefulness of saliva as a sample type
for direct LAMP was investigated. Among the 46 enrolled patients with suspected
VZV infection, 31 patients (67.3%) were positive for the nucleic acid test based on
real-time PCR from skin swab samples. Direct LAMP of skin swabs was positive in 29
(63.0%) of 46 patients. DermaQuick® VZV was positive in 25 (54.3%) of 46 patients.
VZV DNA was detected in only 48.4% of oral swabs with the direct LAMP method.
With real-time polymerase chain reaction (PCR) as the standard for diagnosing
varicella, the sensitivity and specificity of DermaQuick® VZV were 80.7% and 100%,
respectively. The sensitivity and specificity of direct LAMP from skin swabs were
93.6% and 100%, respectively. The sensitivity and specificity of real-time PCR for
DNA extracted from oral swabs were 74.2% and 93.3%, respectively. Thus, oral swab
samples are not suitable for breakthrough varicella diagnosis. Although
DermaQuick® VZV is considered the most convenient point-of-care test for
varicella, its sensitivity and specificity were lower than those of direct VZV LAMP.

28. TREATMENT
• Medication –
• Usually symptomatic medication is given such as anti pyretics, anti histamine etc .
Antivirals can be given to stop viral replication.(Acyclovir)
• Self care-
• Rest adequately
• Calamine lotion and a cool bath with added baking soda, uncooked oatmeal, or
colloidal oatmeal may help relieve some of the itching. Try to keep fingernails
trimmed short and minimize scratching to prevent the virus from spreading to
others and to help prevent skin infections. If you do scratch a blister by
accident, wash your hands with soap and water for at least 20 seconds.
• Nutrition-maintain adequate nutrition and hydration

30. PREVENTION
• The best way to prevent chickenpox is to get the
chickenpox vaccine. Everyone—including
children, adolescents, and adults—should get
two doses of chickenpox vaccine if they have
never had chickenpox or were never vaccinated.

31. VACCINATION
• Varicella vaccine-
• Description-
• Name -Varivax
• Target –Varicella
• Vaccine type – live attenuated vaccine
• Route of administration- sub-cutaneous
• Dose- 0.5ml
• Efficacy-
• One dose of vaccine prevents 95% of moderate
disease and 100% of severe disease. Two doses of
vaccine are more effective than one.

32. HISTORY OF VARICELLA VACCINE
• Michiaki Takahashi developed the live attenuated varicella vaccine in 1974 . This was
the first, and is still the only, herpesvirus vaccine. Early studies showed promise, but
the vaccine was rigorously tested on immunosuppressed patients because of their
high risk of fatal varicella; vaccination proved to be lifesaving. Subsequently, the
vaccine was found to be safe and effective in healthy children. Eventually, varicella
vaccine became a component of measles mumps rubella vaccine, 2 doses of which
are administered in the USA to ~90% of children. The incidence of varicella has
dropped dramatically in the USA since vaccine-licensure in 1995. Varicella vaccine is
also associated with a decreased incidence of zoster and is protective for
susceptible adults. Today, immunocompromised individuals are protected against
varicella due to vaccine-induced herd immunity. Latent infection with varicella zoster
virus occurs after vaccination; however, the vaccine strain is impaired for its ability to
reactivate.

33. Vaccination dose Vaccination age
1st dose 12-15 months of age
2nd dose 4-6 years of age (may be given earlier, minimum
gap of 3 months after the 1st dose )
People not been vaccinated or varicella vaccine for
adult
People of 13 years or older should get 2 doses at
least 28 days apart

34. CONTRAINDICATION
• Immunosuppression
• Pregnancy
• moderate or severe acute illness
• Hypersensitivity to vaccine component or following a prior dose

35. SIDE EFFECTS
1.Injection site complaints-
• Pain
2.Fever
3.Rash-(3-4% people)
• -rash may be maculopapular than vesicular
• -average 5 lesions
4.Systemic reactions are not common.

36. VARICELLA ZOSTER
IMMUNOGLOBULIN
Varicella-zoster immune globulin (VZIG) was first researched in the 1960s and prepared from
patients recovering from varicella-zoster.
• Varicella-zoster immune globulin should be a consideration for post-exposure prophylaxis in
individuals who have a high risk of developing severe disease, lack evidence of immunity, or
are ineligible for the vaccination. The CDC recommends varicella-zoster immune globulin
either immediately or up to 10 days following exposure for maximal efficacy for the
following patient groups:
• Immunocompromised patients with no evidence of immunity
• Mothers of newborn infants who have signs and symptoms of varicella around the time of
delivery (i.e., five days before delivery and up to two days after); VZV transmission can be
transplacental (in utero) or can happen during a contact perinatally or postnatally.
• Premature infants hospitalized or born at ≥28 weeks of gestation whose mothers lack
evidence of immunity to varicella.
• Premature infants hospitalized or born at <28 weeks of gestation or weighing <1000g at
birth, even if their mothers have evidence of immunity to varicella.
• Pregnant women lacking evidence of immunity

37. OTHER PREVENTIVE MEASURES
• The spread of chicken pox can be prevented by isolating affected individuals.
• Contagion is by exposure to respiratory droplets, or direct contact with lesion,
within a period lasting from three days before the onset of rash, to four days after
the onset of rash.
• The spread of chickenpox can also be prevented by practicing good hygiene and
washing your hands frequently.
• The chickenpox virus is susceptible to disinfectants, notably chlorine
bleach (i.e., sodium hypochlorite). Like all enveloped viruses, it is sensitive to drying,
heat and detergents.

38. INTENTIONAL EXPOSURE
• Because chickenpox is usually more severe in adults than it is in children, some
parents deliberately expose their children to the virus, for example by taking them
to "chickenpox parties". Doctors say that children are safer getting the vaccine,
which is a weakened form of the virus, than getting the disease, which can be fatal
or lead to shingles later in life.

39. COMPLICATIONS OF VARICELLA
• Serious complications from chickenpox include:
• Herpes zoster/ shingles
• Bacterial infections of the skin and soft tissues in children, including Group A streptococcal
infections
• Infection of the lungs (pneumonia)
• Infection or swelling of the brain (encephalitis, cerebellar ataxia)
• Bleeding problems (hemorrhagic complications)
• Bloodstream infections (sepsis)
• Dehydration
• Congenital varicella syndrome in neonates of varicella affected mother
• Reye’s syndrome in patients taking aspirin

41. PEOPLE AT RISK OF COMPLICATIONS
• People who may get a serious case of chickenpox and may be at high risk for complications
include:
• Infants
• Adolescents
• Adults
• People who are pregnant
• People with bodies that have a lowered ability to fight germs and sickness (weakened
immune systems) because of illness or medications, for example,
• People with HIV/AIDS or cancer
• Patients who have had transplants, and
• People on chemotherapy, immunosuppressive medications, or long-term use of steroids.