From May 2009 through June 2011, investigators from Antelope Valley (AV) and West Philadelphia Varicella Active Surveillance Project (VASP) conducted this matched case-control study in collaboration with the Centers for Disease Control and Prevention (CDC). Institutional review boards at all 3 participating institutions approved the study protocol. The target populations for case and control subject recruitment were residents aged 1 to 18 years from AV and Philadelphia. AV spans approximately 2,200 square miles of northern Los Angeles County and has a population of over 370,000 residents.²¹ Philadelphia is a large and densely-populated metropolis with 1.5 million residents.²² During the study, the majority of AV residents <20 years of age were either Hispanic (51%) or non-Hispanic white (30%).²¹ Among those of same age in Philadelphia, 48% were non-Hispanic black, 28% were non-Hispanic white, and only 16% were Hispanic.²²

In AV and Philadelphia, varicella cases were identified prospectively using population-based active surveillance methods. Over 300 participating community-based sites (e.g., schools, healthcare provider offices, etc.) in each surveillance area reported suspected varicella cases or informed project staff that no cases occurred at their facility biweekly.^{15, 23} During the 2010-2011 academic year, active surveillance was expanded from West Philadelphia to include an additional 232 schools that were located in other areas of Philadelphia. Eligible case subjects in Philadelphia were also identified through citywide passive surveillance. All case reports were investigated using the standardized VASP questionnaire.^{15, 23}

Following investigation, a case subject was defined as an individual residing in AV or Philadelphia with no prior history of varicella and acute onset of a diffuse maculo-papulovesicular rash or for previously vaccinated persons, modified maculopapular rash with few or no vesicles that a medical provider definitively diagnosed as varicella without any other apparent cause.^{15, 24} During May 2009 through July 2010, enrollment was limited to children aged 5 to 14 years with laboratory confirmation of varicella-zoster virus (VZV) by Polymerase Chain Reaction (PCR) testing. During August 2010 through June 2011, enrollment was expanded to include persons 1 to 18 years of age with laboratory and/or clinical diagnosis of varicella by a healthcare provider. Laboratory confirmation was expanded as well to include positive VZV-specific PCR, Direct Fluorescent Antibody (DFA) assay, or culture results.

Control subjects were selected from the Kaiser Permanente Southern California membership database and the Philadelphia Department of Public Health's Kids Immunization Database/Tracking System registry in AV and Philadelphia, respectively. Kaiser Permanente Southern California, an integrated health care system, provides comprehensive health services to 30% of AV residents aged 1 to 19 years; vaccine administration data for its members is stored in the Kaiser Immunization Tracking System and includes information on vaccine doses administered by providers inside and outside the Kaiser network or verified through school or provider records for vaccinations given prior to enrollment. Kaiser Permanente also was an active surveillance reporting site in AV. The Philadelphia Department of Public Health immunization registry has utilized health department birth records and vaccine administration reporting from healthcare providers to establish and maintain immunization records for all children aged ≤6 years in Philadelphia since 1995. The registry expanded to include children aged ≤18 years in 2007.

For each varicella case identified that met study inclusion criteria, we selected potential controls using incidence density sampling by extracting age-matched (+2 years) records for all children from the pool of eligible controls aged 1 to 18 years who did not have a prior varicella history documented in historic surveillance data or immunization registry.²⁵ A two-year age range for controls was chosen, since the routine 2-dose varicella vaccination recommendation spans ages 4 to 6 years.¹³ Moreover, since routine 1-dose coverage reached higher levels (>80%), protection from 1-dose varicella vaccination appears to remain consistent during the first few years after vaccination.¹³ Between 5 and 60 potential control subjects were randomly selected from the corresponding control pool for each incident case. In order to be able to analyze VE among the age groups for which the first and second doses of varicella vaccine are recommended, controls selected for cases aged 1 to 3 years had to be <4 years of age and controls selected for cases ≥4 years of age had to be ≥4 years of age. Study staff sent a study invitation letter and contacted parents or guardians of eligible control subjects via telephone. The first 3 eligible respondents who consented to participate comprised the controls for each incident case. Recruited controls were eligible to be controls for subsequent incident cases, and if s/he developed varicella at a later time point, were eligible for the study as a case subject.

Study staff obtained verbal consent and collected data from a parent or guardian of each subject by telephone using a standard questionnaire. Given limited study resources, we did not recruit cases and controls with non-English-speaking parents or guardians who could not provide consent due to the language barrier. The questionnaires captured information on demographics, varicella vaccination history, recent VZV exposures, underlying medical conditions, and use of immune suppressing medications. The case questionnaire, which has been described previously, included additional disease-specific questions and standardized prompts to obtain number of lesions.²³ VASP staff scheduled home visits to collect lesion specimens from eligible cases reported before their rashes had resolved. The CDC National VZV Laboratory performed PCR testing^{26, 27} on lesion specimens collected from suspected varicella cases. For a few cases, VZV-specific testing was conducted by hospital or commercial reference laboratories. Participating families received a $10–$20 gift card after completion of study-related activities, and AV healthcare providers were offered a $20 gift card for every case reported with lesion specimens collected.

For case and control subjects, varicella vaccination administration dates were collected from the registries used for control selection, parental records, and the subject's healthcare provider. Study staff made efforts to validate vaccination information for all subjects with the immunization registry or healthcare provider records. If a discrepancy existed between these two sources, the source with the most complete information (i.e., highest number of doses) was used. We considered 1-dose varicella vaccination to be valid when given 4 days before a child's first birthday or later. Second-dose varicella vaccination was considered valid when administered ≥4 weeks after the first dose. The last dose also needed to be given >42 days before rash onset for cases (breakthrough varicella) or the matched incident case's onset for controls. Those given doses within 42 days were excluded.

For our main analysis, we combined data from both sites, since varicella vaccine coverage and risk for breakthrough varicella have not differed among socio-demographic subgroups,^{13, 28} and the direction of estimates from each site were similar. We used two case definitions for varicella: clinically-diagnosed and laboratory-confirmed. Varicella severity was categorized based on the number of lesions reported as mild (<50 lesions), moderate (50–500 lesions) or severe (>500 lesions). The following VE estimates were calculated to examine protection against any varicella or moderate/severe disease alone (≥50 lesions): incremental 2-dose VE (2 doses vs. 1 dose), overall 2-dose VE (2 doses vs. unvaccinated), and overall 1-dose VE (1 dose vs. unvaccinated). All VE estimates were calculated using Greenwood and Yule's formula: 1-relative risk (RR).¹⁹ In our study, RR refers to the risk of developing varicella among the subgroup with the higher number of varicella vaccine doses compared with the subgroup with fewer or no doses and was estimated with an odds ratio (OR) from conditional logistic regression to account for the matching variable (age). We were able to adjust for other potential confounders when examining VE against clinically-diagnosed disease using the combined site data. Changes in VE by time since vaccination (rash onset date minus the date of most recent varicella vaccination) were calculated using previously described methods.²⁵ The distribution of categorical or continuous variables between cases and controls were examined with Mantel-Haenszel chi-square test, Fisher's exact test, Mann–Whitney U test, or Kruskal-Wallis test where appropriate. All analyses were conducted in SAS 9.3 (SAS Institute, Cary, NC).

A total of 125 clinically-diagnosed varicella cases and 408 matched controls were enrolled. Of the 44 (35.2%) cases that had lesion specimens tested for VZV, 29 were laboratory-confirmed cases (all PCR positive), 11 were PCR negative (median lesion collection day: 5 [range: 2–21]), 2 had inadequate specimens, and 2 were culture negative. The median age of clinically-diagnosed cases was 2.1 years among those aged <4 years and 9.5 years among those aged ≥4 years (Table 1). For each case, 2 to 7 matched controls (median=3) were recruited after approaching a median of 5 (range: 5–15) and 29 (range: 5–60) eligible individuals in AV and Philadelphia, respectively. The distribution of demographic characteristics did not differ significantly between clinically-diagnosed case and control subjects, except daycare attendance among those aged ≥4 years (P=0.03). Most controls aged ≥4 years from AV and Philadelphia had received at least one dose of varicella vaccine (98.8% vs. 95.5%, P=0.05), and the majority in each site had received 2-doses (78.5% vs. 83.6%, P=0.31). While the proportion of vaccinated cases aged ≥4 years from each site was similar (91%–92%), the proportion of cases aged ≥4 years who were 2-dose recipients was slightly lower in AV than Philadelphia (41.1% vs. 59.5%, P=0.08).

Among clinically-diagnosed cases ≥4 years of age, rash severity and characteristics differed significantly by vaccination status with the majority of breakthrough cases reporting mild and mostly maculopapular rashes while most unvaccinated cases had 50–500 lesions that were mostly vesicular (Table 2). There was no severe varicella among 2-dose cases. Only two cases had >500 lesions; both were otherwise healthy adolescents, of whom, one was a 1-dose recipient and the other unvaccinated. None of the cases were hospitalized due to varicella or developed complications of varicella. Among breakthrough cases aged ≥4 years, 2-dose cases were more likely to have rashes that resolved in <1 week (P=0.01) and were less likely to have vesicular rashes (P=0.01) than 1-dose cases. Presence and duration of fever did not differ significantly between breakthrough and unvaccinated cases.

Among all unvaccinated and 1-dose participants, the effectiveness of 1-dose of varicella vaccine compared with no vaccine was 75.6% [95% confidence interval (CI): 38.7–90.3%] against all clinically-diagnosed varicella and 78.1% (95% CI: 12.7–94.5%) against moderate/severe disease (Table 3). The effectiveness of 2 doses of varicella vaccine compared to no vaccine among subjects aged ≥4 years was 93.6% (95% CI: 75.6–98.3%) against all clinically-diagnosed varicella and 97.9% (95% CI: 83.0–99.7%) against moderate/severe varicella. The incremental effectiveness of 2-dose varicella vaccination compared with 1-dose among participants ≥4 years of age was 87.5% (95% CI: 74.9–93.7%) in preventing any clinically-diagnosed varicella and 94.1% (95% CI: 72.4–98.8%) in preventing moderate/severe clinically-diagnosed disease.

VE estimates were higher but not significantly in AV than Philadelphia. Among subjects aged ≥4 years from AV, 2-dose VE and incremental VE against any clinically-diagnosed varicella were 98.4% and 92.4%, respectively. In Philadelphia, 2-dose VE and incremental VE among subjects ≥4 years old were 92.7% and 79.8%, respectively. Two-dose VE estimates did not differ significantly between sites (P=0.20); however, the small number of unvaccinated cases (≤5) and controls (≤6) may have led to unstable VE estimates by site.

Among the 26 laboratory-confirmed cases aged ≥4 years and their matched controls, 2-dose varicella VE was 95.9% (95% CI: 23.2–99.8%), and incremental VE was 97.3% (95% CI: 88.9–100%). Because data were sparse, we could not assess effectiveness of 1-dose of varicella vaccine against laboratory-confirmed varicella.

Among 2-dose varicella vaccine recipients, there was no association between time since receiving dose 2 and breakthrough varicella (P=0.17, Table 4). However, those who received the second dose after 6 years of age were 60% less likely to have breakthrough varicella than those who had received the second dose at an earlier age (P=0.009). A longer time interval between receiving 1- and 2-dose varicella vaccine (>5 vs. ≤5 years) was associated with lower odds of developing breakthrough varicella (OR=0.5, P=0.03, Table 4). Subjects receiving dose 2 after 6 years of age were older than those receiving the second dose varicella vaccine earlier (12.7 vs. 7.0 years, P<0.001) as were subjects with a time interval between 1- and 2-dose varicella vaccine > 5 years compare with those having a shorter interval between doses (13.0 vs. 7.4 years, P<0.001).