1. Clinical Presentation and Vaccination Status of Laboratory Confirmed PCR-Positive and PCR-Negative Varicella Cases: A Comparison
Karen Kuguru, MPA
Varicella Active Surveillance Project (VASP)
Los Angeles County Department of Public Health
Good Afternoon
My name is Karen Kuguru
I am with the Los Angeles County Department of Public Health
My presentation is a comparison of the Clinical Presentation and Vaccination Status of Laboratory Confirmed PCR-Positive and PCR-Negative Varicella Cases
VASP is funded under a cooperative agreement with
the Centers for Disease Control and Prevention (CDC)

2. Background (1)
Antelope Valley (AV) Varicella Active Surveillance Project (VASP) is one of two national sites conducting active surveillance for varicella and herpes zoster since 1995
Varicella incidence in AV decreased by 97% from 1996 to 2010
10.3 cases per 1000 in 1996 to 0.3 cases per 1000 population in 2010
BACKGROUND
Just to give you a little background information,
Antelope Valley Varicella Active Surveillance Project which I will refer to as AV VASP
is one of two national sites conducting active surveillance for varicella and herpes zoster since 1995
VASP found that Varicella incidence in the AV decreased by 97% from 1996 to 2010
from 10.3 cases per 1000 population in 1996 to 0.3 cases per 1000 population in 2010
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3. Background (2) Diagnosis of varicella is increasingly challenging:
Most varicella cases have prior varicella vaccination
Milder clinical presentation and fewer incident cases
PCR testing provides a more definitive approach for diagnosing varicella disease
PCR testing of skin lesions of varicella zoster virus (VZV) DNA is regarded as the most sensitive and specific method*
100% Sensitivity and Specificity estimates for macular/ papular lesions
*Leung, et al Clinical Inf Dis 2010; 51 (1):23-32
BACKGROUND
Diagnosis of varicella has become increasingly challenging, because most varicella cases have prior varicella vaccination.
This causes the disease to have a milder clinical presentation and fewer incident cases.
PCR testing provides a more definitive approach for diagnosing varicella disease.
It is regarded as the most sensitive and specific method,-
With 100% Sensitivity and Spe-ci-fi-city estimates for macular/ papular lesions collected within 5 days of rash onset.
PCR
-POLYMERASE CHAIN REACTION
- PCR procedure where the DNA of a sample is amplified and replicated to create a large sample then checked to see if the Varicella Zoster Virus can be detected.
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4. Study Objectives
Describe the proportion of varicella cases with PCR testing from 2003 to 2010
Describe PCR testing in varicella cases by age-group
Describe the time to specimen collection in varicella cases with PCR-testing
Describe the clinical presentation of varicella cases with PCR-testing
Describe varicella vaccination status of varicella cases with PCR testing
STUDY OBJECTIVES
My study objectives are; (PAUSE)
Describe the proportion of varicella cases with PCR testing from 2003 to 2010
Describe THEIR PCR testing BY AGE-GROUP
Describe THEIR time OF RASH ONSET to specimen collection
Describe THEIR clinical presentation
Describe THEIR vaccination status

5. Methods: Case Definition
Verified varicella case
An illness with an acute onset of a diffuse papulovesicular rash without other known cause
Diagnosed by a healthcare provider (HCP), school nurse or parent 
Completed case report
Resident of AV
All verified varicella cases from with and without PCR testing were included in analysis
METHODS
Moving on to the Methods:
A Verified varicella case is
An illness
with an acute onset of a
diffuse papulo-vesicular rash
without other known cause
Diagnosed by a healthcare provider, school nurse or parent 
It has a completed case report
And the case is a resident of the AV.
All verified varicella cases from ,
with and without PCR testing,
are included in this analysis.
Papulo-raised red bumps
Vesicular- fluid filled

6. Methods: Data Collection
Standardized telephone interview conducted with each varicella case or their parent/guardian
If parent/guardian is not available for interview, medical charts are used for verification of diagnosis
Vaccination information is confirmed by immunization record from parent, school or HCP
METHODS: DATA COLLECTION
Say data collection…….
A Standardized telephone interview is conducted with each varicella case or their parent or guardian
If the parent or guardian is not available for interview, medical charts are used for verification of diagnosis
Vaccination information is confirmed by immunization records from parents, schools or healthcare providers

7. Methods: PCR Testing of Skin Lesions
Schools are encouraged to report suspected cases immediately
VASP staff encourage HCP to follow-up and complete PCR testing
Beginning 2009, VASP staff made home visits to collect skin lesion specimens
Specimens are sent to CDC’s National Varicella Zoster Virus (VZV) Laboratory for PCR testing
METHODS: PCR TESTING OF SKIN LESIONS
Schools are encouraged to report suspected cases immediately.
We encourage cases to visit their healthcare providers so that PCR testing can be completed
Beginning 2009,
VASP staff,
started making home visits to collect skin lesion specimens,
if not previously done by Healthcare Providers
Specimens are sent to
CDC’s National Varicella Zoster Virus
Laboratory for PCR testing

8. Methods: Interpreting PCR Lab Results
PCR-positive varicella case
VZV positive
Actin positive or negative
PCR-negative varicella case
VZV negative
Actin positive
Inadequate specimen
Actin negative or missing or unknown
METHODS: INTERPRETING PCR LAB RESULTS
Say – Interpreting lab results
There are 3 possible results from skin lesion testing. PCR+, PCR- and inadequate specimens
PCR-POSITIVE VARICELLA CASES
If a specimen is VZV-Positive and either
Actin Positive or Negative,
it is regarded as true positive case because the viral DNA was detected.
PCR-NEGATIVE VARICELLA CASES
If a specimen is VZV-Negative and
Actin-Positive,
it is regarded as a true negative case.
INADEQUATE SPECIMEN CASES
If a specimen is both VZV-Negative and
Actin-Negative,
It is impossible to determine whether it’s really a negative case,
so its is classified as
Inadequately collected. 24/234(10 %)
Also included in the inadequate specimens,
were ANY specimens
that had a missing or unknown ACTIN result. 13/234(6%)

9. Results: Completion of PCR Testing Among Verified Varicella Cases, N= 2595, 2003 -2010
RESULTS: VARICELLA CASES AND PCR TESTING
Moving on to results,
From January 1ST through December 31ST there were 2595 varicella cases reported to VASP  
Of this, 234 which was 9%, had PCR testing
When we look at the graph,
we see that between 2009 and 2010,
PCR lab-tested cases increased from 10% to 20%
Over the last 2 years,
VASP has worked with healthcare providers,
to expand PCR laboratory testing
And started home visits
to collect specimens from suspect cases.

10. Results: PCR Testing Results by Year, N=233, 2003-2010
Say…..PCR testing by year……..
84% of PCR tests conducted during this study period,
had adequate specimens
As we can see in this graph,
2004 had the most PCR testing and
the most PCR-positive cases of the study period
This was because of a dramatic upswing of school outbreaks in the AV,
with 25 documented outbreaks that year
From 2006 to 2009,
as the number of reported cases decreased,
so did the number of pcr-tested cases

11. Results: Overall PCR Test Results, N=233, 2003-2010
Total PCR-positive cases: 170 (73%)
VZV strain type:
Wild type: 164 (96.5%)
Vaccine strain 5 (2.9%)
Unknown 1 (0.6%)
Total PCR-negative cases: 26 (11%)
Inadequate specimen cases: 37 (16%)
RESULTS: OVERALL PCR TESTING
73% of total PCR tested cases were PCR-positive
Of those,
96% were of the Wild type strain
3% were the vaccine strain and
Under 1% had an Unknown strain type
11% of total PCR tested cases were PCR-Negative and
Inadequate specimens made up 16% of total PCR tested cases
Inadequate Specimen
VZV-Negative and Actin-Negative 24/233(10%) of total specimens
PCR Actin results that were missing or classified as indeterminate or unknown 13/234(6%)
Wild type – varicella that is not associated with the vaccine, because it occurred more than 42 days after vaccine was given

12. Results: PCR Testing by Age Group, N= 233, 2003-2010
Say….PCR testing by Age-group
The greatest proportion of PCR-positive cases and Inadequate Specimen Cases,
were in the 10-14
then the 5-9 age groups
The greatest proportion of PCR-negative cases ,
were in the 5-9 and 1-4 age-groups
These age-groups represent the peak years of “breakthrough” varicella
which are the age groups that are targeted for vaccination
Breakthrough disease- a case of wild type varicella that occurs in a person more than 42 days after vaccination following exposure of wild type virus
- Rash onset more than 42 days after vaccination

13. Results: Time of Specimen Collection by PCR Result, N=233, 2003-2010
…….jump right in…………
We looked at the number of days from rash onset to specimen collection.
We found that 75 % of all PCR tested cases,
were collected within 5 days of rash onset
which is the recommended time for best PCR results
77% of PCR-positive cases were collected within 0 -5 days
While 11% were collected within 6 to 10 days
and 13% were collected over 10 days of rash onset

14. Results: Lesion Grading of Varicella Cases with PCR Testing, N=233, 2003-2010
Say…lesion grading…..
For PCR-positive cases
42% had <50 lesions
42% had 50 to 249 lesions
10% had lesions
2.4% had more than 500 lesions
Cases with more lesions were more likely to be PCR positive cases.
Which ones were breakthrough varicella
Which ones were unvaccinated

15. Results: Clinical Characteristics of Varicella Cases and PCR Status, N=233, 2003-2010
PCR –POSITIVE
N=170
PCR –NEGATIVE
N=26
INADEQUATE SPECIMEN
N=37 %
Macular/Papular 58 81 68
Vesicular 39 15 30
Fever Before Rash 22 8 32
Fever After Rash 42 19 27
…..say clinical presentation……
A greater proportion of PCR positive cases had fever and vesicular lesions than PCR negative.
PCR –negative cases had the highest proportion of macular/papular lesions
All groups had greater proportions of macular/papular lesions than vesicular lesions

16. CASES WITH ADEQUATE PCR SPECIMEN* ALL OTHER VARICELLA CASES
Results: Clinical Characteristics of Varicella Cases With and Without PCR Testing, N=2595,
CLINICAL
DESCRIPTION 
CASES WITH ADEQUATE PCR SPECIMEN*
N=196
ALL OTHER VARICELLA CASES
N=2399 %
Macular/Papular 61
Vesicular 36 35
Fever Before Rash 20 26
Fever After Rash 39 33
This table compares the clinical characteristics of PCR lab-confirmed varicella cases,
With ALL OTHER verified varicella cases,
that WERE REPORTED to VASP
but were NOT PCR lab tested
-inadequate specimens are included in the ‘all other varicella cases’
-We found that the characteristics of PCR lab confirmed cases,
-were comparable to cases that were verified by clinical characteristics,
- which is the method that most health departments USE TO IDENTIFY chickenpox disease.
*37 cases excluded with inadequate specimens

17. Results: Varicella Cases by PCR and Varicella Vaccination Status, N=230*, 2003-2010
PCR- Positive
N=168*
PCR-Negative
N=26
Inadequate
Specimens
N= 36**
Total
N=230
n (%)
n ( %)
Unvaccinated
68 (41)
1 (4)
13 (36)
82(36)
1 Dose
96 (57)
22 (85)
20(56)
138(60)
2 Doses
4 (2)
3 (11)
3(8)
10(4)
….vaccinations……..
More vaccinated cases than unvaccinated had PCR testing (64%)
60% of all cases had received one varicella vaccine dose
PCR-positive cases had a larger proportion of unvaccinated cases than PCR-negative cases
WHILE 85% of PCR-negative cases had received one varicella vaccine and 11% had received both.
PCR-positive lab confirmation was more likely for unvaccinated than vaccinated cases???
*2 PCR-positive cases with unknown vaccination status
**1 Inadequate specimen case with unknown vaccination status

18. Study Limitations
Most cases were reported by schools and represent school age children
the results may not be generalized to older varicella cases
Varicella cases with PCR testing had HCP follow-up for testing
Results may be less representative of milder varicella cases that did not seek HCP follow-up
Challenging to collect specimens within 5 days of rash onset with biweekly active surveillance
Moving on to our study limitations…………………..
Because most OF OUR cases were reported by schools,
our study represents school age children,
And so our results may not be generalized to older varicella cases
Most PCR tested cases saw a healthcare provider for testing,
which makes our results less representative
of milder varicella cases that did not visit a healthcare provider.
It is challenging to collect specimens within 5 days of rash onset with biweekly active surveillance

19. Summary
Only 9% of the total varicella cases were PCR tested over the study period
PCR testing increased from 0.7% to 20% from 1993 to 2010
PCR-positive varicella cases had a clinical presentation more closely resembling “classic” varicella vs. PCR-negative cases
At least 75% of PCR- positive cases were collected within 5 days of rash onset
However, PCR- positive cases where confirmed when specimen was collected 10 days after rash onset
Here’s the summary of our findings………………………..
Only 9% of the total varicella cases were PCR tested over the study period.
PCR testing increased from 0.7% to 20% from 1993 to 2010.
PCR-positive cases had a clinical presentation,
that more closely resembled “classic” varicella than PCR-negative cases.
At least 75% of PCR- positive cases were collected within 5 days of rash onset.
However, PCR- positive cases were still confirmed
when specimen was collected 10 days after rash onset

20. Conclusions
PCR- based testing is especially important in vaccinated varicella cases
Varicella infection was confirmed in 4 cases with two doses of varicella vaccine
Lab testing is increasingly important
Increasing vaccination coverage
Modified clinical presentation
And now to our conclusions…………
PCR- based testing is especially important in vaccinated varicella cases.
Varicella disease has been confirmed in 4 cases who had received both doses of the varicella vaccine.
Lab testing is very important due to increasing vaccination coverage
which has modified the clinical presentation of varicella disease.
THANKYOU VERY MUCH FOR YOUR TIME AND I WELCOME ANY QUESTIONS

21. Acknowledgements
Varicella Active Surveillance Project, Los Angeles Department of Public Health
Laurene Mascola, MD, MPH, VASP Principal Investigator
Rachel Civen, MD, MPH, VASP Co-Principal Investigator
Christina Jackson, MPH, Project Manager
Amanuel Hussien, MSc, MSW, Research Analyst
Michael Borquez, Clerical support staff
Michelle Armijo, Student Worker
Division of Viral Diseases, NCIRD, Centers for Disease Control and Prevention
Stephanie Bialek, MD, MPH
Scott Schmid, MD, Ph.D
Adriana Lopez, MHS

22. Q & A

23. Study Site: Antelope Valley (AV), California
Geographically circumscribed
Located in northeastern corner of Los Angeles County (LAC)
~ 2,200 square miles with total population ~ 366,000
Mean birth cohort: 5,900 infants
The Antelope valley in California is located in the northern Los Angeles County and the southeastern portion of Kern County and western tip of the Mojave Desert. It is high desert community covers 2,200 square miles with total population 366,000. It is assumed that most people that live in the Antelope Valley get their medical care locally and children attend school in Antelope Valley.
From the mean birth cohort was 5,900 infants.
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24. Background : Varicella Cases and Incidence Rates by Lesion Grading (N=2595, 2003-2010)
Clinical presentation of varicella disease has become milder over the years.
In 1995, 1035(35.3%) cases reported <50 lesions compared with 318(49.3%) in 2004 and 52(57.7%) in 2010.
Moderate varicella of lesions has decreased from 1341(45.7%) in 1993, 253(39.2%)in 2004 and 28(29.5%) in 2010.
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25. Methods: Collection of Specimens for PCR Testing (4)
Using a sterile swab, rub the lesion vigorously enough to ensure that skin cells or fluid are collected.
Samples should be taken from ≥2 lesions to ensure adequate specimen for PCR testing

26. Results: Reporting Sources of PCR Tested Cases (N=233, 2003-2010)
In total, 233 varicella cases had specimens PCR based testing
Cases Reported by:
Schools: 113 (49%)
Elementary:84(36%)
Middle-High: 25(11%)
Childcare: 2(1%)
High school: 2(1%)
HCP: 106(45%)
Household: 7(3%)
Other: 7(3%)
Varicella Diagnosed by:
HCP: 193(83%)
Parent/Guardian: 40(17%)

27. ALL OTHER VARICELLA CASES
Results: Age Group and Completion of PCR Testing compared to Varicella Cases without PCR Testing, N=2595,
 Age (yrs)
CASES WITH
ADEQUATE PCR SPECIMEN
N=196*
ALL OTHER VARICELLA CASES
N=2399
n (%)
<1
10(5.1)
97(4.0)
1 to 4
29(14.8)
338(14.1)
5 to 9
68(34.7)
903(37.6)
10 to 14
79(40.3)
733(30.6)
15 to 19
7(3.6)
161(6.7)
>19
3(1.5)
167(7.0)
Age of cases with PCR testing was comparable with the all other varicella cases that were not PCR tested
There was no statistically significant results within groups by age of those with PCR testing compared to those without PCR testing- possible due to small sample size of cases
PCR tested cases were similar in age to varicella cases without PCR testing
*37 inadequate specimens excluded
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28. Vaccine Doses and Strain Type, N=170
PCR-positive
N=170
N(%)
One dose 96
Wildtype Strain
92(54)
Vaccine Strain
4(2)
Two doses 4
3(2)
1(1)
Unvaccinated 68
67(40)
Unknown strain
Unknown vaccination 2
Wildtype
Wildtype =

29. Vaccination Status vs. Lesion Grading, PCR+ and PCR-
PCR-Positive
N=170
1 dose
2dose
Unvaccinated
Unknown
vaccination
<50
53(31)
3(2)
14(8)
2(1)
50 to 249
34(20)
1(1)
36(21)
4 (2)
13(8)
>500
missing
4(2)
PCR negative
N=26
1 dose
2dose
Unvaccinated
Unknown
vaccination
<50
18(69)
2(7)
50 to 249
3(12)
1(4)
>500
Unvaccinated had the most lesions

30. Breakthrough Varicella Vs. Days after vaccination, N=148
PCR-positive
PCR-negative
Total
0-42
7(5)
3(2)
10(7)
>42
101(68)
37(25)
138(93)
Only vaccinated case – missing all unvaccinated
How long after vaccination did case come down with breakthrough varicella
All given VV for the study is =148
True wildtype cases = = 138
93% of cases who were vaccinated, had rash occurring more than 42 days after - vaccination
7% of vaccinated cases had rash 0-42 days after vaccination-possibly vaccine strain