1. The presenter has no conflicts of interest to disclose.
Laboratory Confirmation and Evaluation of Case Definitions for Diagnosing Breakthrough Varicella Infections
Dana M. Perella1
B Watson1, K Heath1, D Robinson1,
S Schmid2, CV Spain1
Varicella Active Surveillance Project
1Philadelphia Department of Public Health
2Centers for Disease Control and Prevention
Good morning.
Today, I will present findings that describe some of the challenges to diagnosing varicella in the post-vaccine-licensure era.
Before I begin, I would like to acknowledge my co-authors from the Varicella Active Surveillance Project at the Philadelphia Department of Public Health and CDC.
This research was supported by cooperative agreement U66/CUU , National Immunization Program, Centers for Disease Control and Prevention.
The presenter has no conflicts of interest to disclose.

2. Varicella (Chickenpox) Vaccine
Licensed for use in the US in March 1995
Initially recommendations for healthy susceptible persons:
12 months to 12 years: 1 dose*
>13 years: 2 doses given one month apart
Efficacy of one-dose regimen from clinical trials
70% to 90% protection any form of varicella disease
95% against severe varicella disease
The varicella or chickenpox vaccine was licensed for use in the US in 1995.
Initially, one-dose of the varicella vaccine was recommended for healthy susceptible children 12 months to 12 years and a two doses given one month apart were recommended for those 13 years of age and older.
From the clinical trials, the varicella vaccine was shown to provide 70 to 90% protection against any form of varicella and 95% protection against severe disease. Most evaluations of varicella vaccine effectiveness since licensure have been consistent with the clinical trial findings.
*In June 2006, ACIP revised the recommendations for use of the varicella vaccine to include a second dose for those 4 years of age and older who have previously received one dose..

3. Classic Varicella vs. Breakthrough
Unvaccinated Case
Breakthrough Case
This slide compares classic varicella in an unvaccinated child to breakthrough varicella in a previously vaccinated child.
Classic varicella in an unvaccinated person has a distinct presentation and typically involves a generalized vesicular rash consisting of lesions with fever and malaise.
Breakthrough varicella infections are infections due to wildtype varicella-zoster virus (VZV) occurring >42 days after vaccination.
In contrast to classic varicella, most breakthrough varicella are mild with <50 lesions and few or no vesicles. These modified rashes have been described in recipients of both the one-dose and two-dose regimens.
These mild breakthrough infections are easily confused with other rashes including insect bites, cold injury, drug rash, and dermatitis)
lesions
Mostly vesicular
2 - 4 “crops” of lesions
Fever, Malaise
<50 lesions
Atypical appearance
Few or no vesicles
Less contagious

4. Challenges to Confirmation of Varicella Post-Vaccine-Licensure
As vaccine coverage increases, an increase in the proportion of varicella cases that are breakthrough infections is expected.
CDC: Varicella Zoster Virus (VZV) laboratory testing only recommended in special circumstances.
Some challenges to diagnosing varicella in post-vaccine licensure include the increase in the proportion of varicella cases that are breakthrough infections that has occurred as vaccine coverage has increased.
While healthcare providers are diagnosing and reporting fewer patients with any form of varicella, the majority of patients now presenting with varicella-like illness are breakthrough infections.
For purposes of surveillance, CDC only recommends testing of suspected varicella cases during special circumstances (i.e., an outbreak).

5. Objectives
Can breakthrough infections be diagnosed based on clinical and demographic factors alone?
Study Objectives
Estimate the proportion of suspected breakthrough varicella infections confirmed by laboratory testing as VZV infections.
Describe the use of VZV-specific laboratory services to confirm suspected breakthrough varicella infections.
Evaluate potential case definitions for breakthrough infections using combinations of clinical and demographic factors.
These challenges lead us to the question can breakthrough infections be diagnosed based on clinical and demographic factors alone. The specific objectives of our study were to:
Estimate the proportion of suspected breakthrough varicella infections confirmed by laboratory testing as VZV infections.
Describe the use of VZV-specific laboratory services to confirm suspected breakthrough varicella infections.
Evaluate potential case definitions for breakthrough infections using combinations of clinical and demographic factors.

6. Methods Cross-sectional study Suspected case finding
Varicella-reportable disease since 1995
Active Surveillance-West Philadelphia
Passive Surveillance-Outside West Philadelphia
Suspected case investigation interview to collect clinical and demographic information
We used a cross-sectional study design. Since varicella disease has been reportable by law in the City of Philadelphia since 1995, we received suspected varicella case reports included in the study through both active surveillance and passive surveillance. Parents/guardians of all suspected breakthrough varicella cases included in the study were interviewed to collect detailed clinical and demographic information on the suspected case.

7. Inclusion Criteria
Age-appropriate varicella vaccine dosage according to initial recommendations
Rash occurring >42 days from vaccination
Rash onset between 10/1/01 and 12/31/04
Residence not limited to Philadelphia
One or more Varicella-Zoster Virus (VZV)-specific laboratory tests:
Polymerase Chain Reaction (PCR)
Viral Culture
Direct Fluorescence Antibody (DFA)Testing
IgM Enzyme Linked Immunosorbent Assay (ELISA)
(Non-Commercial)
Acute and Convalescent IgG ELISA (Non-Commercial)
To be included in the study, the suspected breakthrough case needed to have the age-appropriate dosage of varicella vaccine according to the initial recommendations. Time from vaccination to rash onset needed to be more than 42 days. Rash onsets must have occurred during our study period which was October 2001 through December We did not limit residence to the city of Philadelphia. ). Included suspect cases must also have received one or more of the following VZV-specific laboratory tests as described in the CDC Manual for Surveillance of Vaccine Preventable Diseases for laboratory confirmation of VZV. Please note, serologic tests for VZV IgM and IgM were included only if they were performed by the CDC’s National VZV Laboratory.

8. Study Definitions Confirmed breakthrough case:
PCR positive wildtype VZV and / or
DFA positive and / or
Culture positive and / or
IgM positive and / or
Acute to convalescent IgG titer increase
>2-fold increase when endpoint titrations performed
>0.350 increase Adjusted Optical Density (OD) Units for others
Negative breakthrough case reports: in absence of another positive result
PCR negative results < day 10 of rash and / or
No acute to convalescent IgG titer rise and / or
Negative/equivocal convalescent IgG results
Indeterminate cases: did not meet criteria for confirmed infection or negative reports
(e.g., PCR specimen inadequate for testing and IgM negative result)
We considered any suspected case with a positive PCR, DFA, Culture, or IgM result to be a true breakthrough case. Also, those with an acute to convalescent IgG titer increase 2 fold or greater when endpoint titrations were performed or IgG titer change >0.350 Adjusted Optical Density units. In absence of a positive result, we considered anyone with the following results to be a negative breakthrough case report: A PCR negative result collect on or before day 10 of rash; No acute to convalescent IgG titer rise; Negative or Equivocal convalescent IgG results.
Anyone not meeting the criteria for a confirmed infection or negative report was considered an Indeterminate case. For example, a suspected case with a PCR specimen inadequate for testing and an IgM negative result would be considered an Indeterminate case.

9. Results: Study Population
411 suspected breakthrough infections included
Median age: 6 yrs (Range: yrs)
Median time from vaccination to rash onset: 4.4 yrs (Range: 48 days yrs)
~3/5 vaccinated between 12 and 17 months
Race Distribution: African American/Black (57.4%), White (28.5%), and Other (14.1%)
HCPs collected acute specimens for 354 (86.1%) case reports.
A total of 411 suspected breakthrough cases were included in our study. Their median age was 6 years and the median time from vaccination to rash onset was 4.4 years. About three-fifths of the case reports were vaccinated before 18 months of age. About 60% of the suspected case reports were African American. Most had acute specimens collected by their healthcare provider during an office visit, while only 14% were collected by health department staff.

10. VZV Laboratory Confirmation
Of the 411 suspected breakthrough case reports, 31.4% (26.9%, 36.1%) were confirmed by laboratory testing as true VZV infections.
Another 45.0% (40.1%, 50.0%) were considered negative breakthrough reports.
23.6% (19.6%, 28.0%) could not be categorized as a confirmed or negative case and fell into the indeterminate category.
All Suspected Cases
n=411

11. PCR Results Results (n=372) 112 (30.1%) were positive for wildtype VZV
187 (50.3%) were negative for VZV,
73 (19.6%) specimens were inadequate for PCR testing
I will now describe the laboratory results by test type. Of the 372 suspected breakthrough varicella cases with PCR testing performed, 30% were positive, 50% were negative, and 20% had specimens inadequate for testing

12. VZV PCR Results and Percent Positive By Day of Rash
This next slide shows PCR Results by day of specimen collection. The bars represent the number of case reports by collection day and result. The red line shows the percentage of cases with PCR positive results. Timing of specimen collection differed significantly by PCR result.
As you can see, all positive results collected by day 10 of rash and 2/3 of the specimens testing positive were collected by day 3, which was slightly earlier than the negative results and inadequate specimens.

13. DFA and Viral Culture Results
DFA (n=7)
3 specimens positive
1 specimen negative
3 specimens were inadequate for testing
Viral Culture (n=1)
Positive for VZV
All specimens for DFA testing and Viral Culture were collected by day 3 of rash.
Only 7 suspected cases had DFA testing performed. 3 had positive results, 1 had a negative result, and 3 specimens were inadequate for testing. The one suspected breakthrough case with a viral culture performed had a positive result. All 8 specimens for dfa testing and viral culture were collected by day 3 of rash.

14. IgM Results Results (n=337)
15 (4.5%) positive
23 (6.8%) equivocal
299 (88.7%) negative
Timing of serologic specimen collection by result differed significantly (P=.009):
Positive: Median-day 7 (Range: 1 to 12)
Equivocal: Median-day 4 (Range: 1 to 9)
Negative: Median-day 3 (Range: 1 to 43)
337 suspected case reports had serologic specimens collected and tested for VZV specific IgM by the CDC National VZV Laboratory. Of those with IgM testing performed, only 4.5% were positive, while 6.8% had equivocal results and the vast majority had negative IgM results.
As you can see, timing of serologic specimen collection differed by IgM result. Those with positive results had a median day of collection that was slightly later (day 7) than those with equivocal or negative results.

15. Acute and Convalescent IgG Results
Endpoint titration results (n=30)
8 (27%) with 2 fold or higher increase
22 (73%) with no change or decrease
IgG Titer Change in Adjusted OD Units(n=48)
9 (19%) with >0.350 increase
10 (21%) with increase  
16 (33%) with no change or decrease
13 (27%) with negative /equivocal convalescent results
Other Convalescent Results
5 (16%) negative/equivocal of 32 not included previously
The following slide includes results for the 52 suspected case reports who had appropriately timed acute and convalescent serologic specimens collected and were tested for VZV specific IgG by the CDC National VZV Lab. Of the 30 suspected breakthrough cases with endpoint titrations performed on their paired serologic specimens, 8 or 27% had a 2 fold or higher IgG increase from the acute to convalescent phase of illness. As for the 48 with acute to convalescent titer change available in OD units, 19% had a >.350 increase from the acute to convalescent phase and were considered confirmed VZV infections. 21% had an increase of indeterminate significance, 33% had not change or a decrease and 27% had negative or equivocal IgG results.
Additionally, 5 case reports not included the previous summary due to inappropriate timing between specimens or lacked an acute serologic specimen had negative or equivocal IgG results and were considered negative breakthrough reports.

16. Clinical and Demographic Variables Evaluated
>50 lesions
Mostly Vesicular Rash
Itching
Scabbing
Rash appearing in crops
Fever
Not enterovirus season (Not July-Sept)
Reported varicella/ herpes zoster exposure
Attending school
Attending daycare
Race other than African American
HCP certain of varicella diagnosis
Rash lasted 5 days or less
The following slide shows the clinical and demographic variables we examined as part of the case definition analysis for breakthrough illness. Please note only those that were confirmed breakthrough infections or negative breakthrough case reports were included in this analysis. Multivariable logistic regression was used to determine the best predictors of laboratory confirmation.

17. Best Predicators of Laboratory Confirmation
Best Set of 5 Predictors
Reported varicella / herpes zoster exposure
Attending school
HCP certain of varicella diagnosis
Scabbing
Race other than African American
The best predictors of laboratory confirmation according to multivariable logistic regression analysis were a reported exposure to varicella or herpes zoster, attending school, having a diagnosis of varicella from a healthcare provider where they were confident the rash was VZV,having a rash that scabbed, having a racial background other than African American
Determinate Results Only:
n=279

18. Case Definition Evaluation: Example 1
Two or more of the following:
VZV Exposure,
HCP Certain,
Scabbing,
Attending School,
Race Other Than AA
Laboratory Confirmed Breakthrough Case
Negative Breakthrough Case
Total
Yes
116
118
234 No 3 42 45
119
160
279
We then examined the performance of the best predicators alone and in combination with one another as case definitions for breakthrough varicella. We compared the potential case definition to the laboratory confirmation result and were then able to calculate the sensitivity and specificity of the case definition. The first example examines a definition based on having 2 or more of the following: a vzv exposure, certain diagnosis, scabbing, attending school or a racial background other than African American. With this definition, 116 case reports were true positives having met the case definition criteria and being confirmed by laboratory testing. 118 were false positives that met the case definition criteria but were not laboratory confirmed. Only 3 were false negatives or case reports that were laboratory confirmed but did not met the case definition criteria. The remaining 42 were true negatives not meeting the case definition criteria and having negative lab results. To calculate sensitivity of the case definition, the number of true positives were divided by the total number of laboratory confirmed cases. As you can see, this definition had a high sensitivity of 97%. To calculate specificity we divide the number of true negatives by the number of negative laboratory results. This definition has a low specificity of 26%. If we use a definition with low specificity we would we have a high number of false positive cases.
Sensitivity =
116
/ 119
= 97%
Specificity = 42
/ 160
= 26%

19. Case Definition Evaluation: Example 2
VZV Exposure,
HCP Certain, Attending School, Race Not AA
Laboratory Confirmed Breakthrough Case
Negative Breakthrough Case
Total
Yes 19 2 21 No
100
158
258
119
160
279
For example 2, we examining the performance of a case definition that includes vzv exposure, a certain healthcare provider diagnosis, attending school, and having a racial background other than African American. To calculate sensitivity we divide the true positives by the total number of laboratory confirmed cases. For the specificity, we take the true negatives divided by the total number of negative breakthrough cases by laboratory testing. As you can see this definition has a low sensitivity, so we are missing many of the laboratory confirmed cases. However, since it has a high specificity the number false positive cases is reduced.
Sensitivity = 19
/ 119
= 16%
Specificity =
158
/ 160
= 99%

20. Case Definition Evaluation: All Combinations
High Sens. Region
High Spec. Region
Sensitivity
This figure plots the Sensitivity and 1-specificity for all possible combinations of the 5 best predictors. Ideally, a case definition should fall into the top left corner where the high sensitivity and high specificity regions over lap. As you can see, none of the potential case definitions based on the different combinations of the 5 best predictors were both highly sensitive and highly specific.
Determinate Results Only:
n=279
1 - Specificity

21. Case Definition Evaluation: Best Performance
High Sensitivity
Any 2
Scabbing
High Sensitivity and
Moderate Specificity
Any 3
Moderate Sensitivity
and High Specificity
Exposure
High Specificity
All 5
Exposure, HCP Certain, School,
Race Not AA 1 2
High Sens. Region 3 4
Sensitivity
High Spec. Region 5
We did find case definitions that were either highly sensitive or highly specific. Examples of the high sensitivity definitions include any 2 of the best five predictors or scabbing alone. Remember with the highly specific definitions we are including most of the laboratory confirmed breakthrough reports; however, many case reports meeting the case definition are false positives and have negative results. Using Any 3 of the best 5 predictors has high sensitivity (86%) and moderate specificity (69%). 6
1 - Specificity
Determinate Results Only:
n=279

22. Conclusions
Proportion of suspected breakthrough cases confirmed as VZV infections was modest.
If offered, HCPs will utilize VZV lab services as part of clinical management for suspected cases.
No clinical case definition was both highly sensitive and specific.
In conclusion, only a modest proportion of suspected breakthrough cases were confirmed as true VZV infections.
Healthcare providers will utilize VZV laboratory services as part of the clinical management of suspected breakthrough cases. However, it is important to provide them with education and training on appropriate specimen timing and technique, so determinate results are produced.
We did not find a clinical case definition for breakthrough varicella that was both highly sensitive and specific.

23. Future Implications VZV Laboratory Testing Available? Yes No
Meets Highly Specific Clinical Definition Criteria?
(e.g., All of the following:
VZV Exposure,
HCP Certain of Diagnosis,
Attending School,
Race Other Than AA)
Meets Highly Sensitive Clinical Definition Criteria?
(e.g., Any 2 of the following:
VZV Exposure,
HCP Certain of Diagnosis,
Scabbing,
Attending School,
Race Other Than AA)
Given the difficulties to the diagnosis of breakthrough varicella, expanded use of VZV laboratory services (preferably PCR testing) should be considered for both clinical management and surveillance purposes. We expect even further declines in varicella disease now that the universal two dose recommendation is being implemented for those 4 years of age and older. If VZV PCR testing is available, a highly specific definition such could be used along with testing. For example, if a suspected case met the case definition including all 5 predicators we considered that case confirmed. Remember, there are fewer false positive cases when we use a highly specific definitions. Those not meeting the highly specific definition or in this case not meeting all 5 criteria would have PCR testing performed. Those with positive results or inadequate specimens would be considered varicella cases and only those with negative results would be excluded as cases. In situations, where testing was not available a highly sensitive definition should used to capture as many true cases as possible. The downside to using a highly sensitive definition is that you will have many false positive cases.
Yes No
Yes No
Varicella
Lab Testing
Varicella
Not Varicella
Confirmed/
Indeterminate
Negative
Varicella
Not Varicella

24. Acknowledgements Participating Healthcare Provider Sites
CDC National VZV Laboratory Staff
CDC Herpes Viruses Team Staff
Some additional acknowledgements include the participating healthcare provider sites
CDC National VZV Laboratory Staff
CDC Herpes Viruses Team Staff