on January 5, 2020 by guest · 60 adolescents and adults (19). In 2005, Td ap, the adult tetanu s-diphtheria-acellular 61 pertussis vaccine that contai ns the same antigenic compone

Pertussis Diagnosis after Vaccination 1

Does Tdap Vaccination Interfere with Serodiagnosis of Pertussis Infection? 1

2

Running Title: Pertussis Diagnosis after Vaccination 3

4

Lucia C. Pawloski1*, Kathryn B. Kirkland2, Andrew L. Baughman1, Monte D. Martin1, 5

Elizabeth A. Talbot2, Nancy E. Messonnier1, Maria Lucia Tondella1 6

7

8

1Centers for Disease Control and Prevention, Atlanta, GA 30333 9

2Dartmouth-Hitchcock Medical Center, Lebanon, NH 03756 10

11

A part of the information was presented at the 9th International Bordetella Symposium, 12

Baltimore, MD, USA, September 30 - October 3, 2010. 13

14

15

*Corresponding Author Contact Information 16

Lucia C. Pawloski 17

Centers for Disease Control and Prevention 18

1600 Clifton Rd. 19

Atlanta, GA 30333 20

Telephone: 404-639-4506 21

Fax: 404-639-4421 22

Email address: [email protected] 23

Copyright © 2012, American Society for Microbiology. All Rights Reserved.Clin. Vaccine Immunol. doi:10.1128/CVI.05686-11 CVI Accepts, published online ahead of print on 25 April 2012

on February 2, 2020 by guest

http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


ABSTRACT 24

Background. An IgG anti-pertussis toxin (PT) enzyme-linked immunosorbent assay 25

(ELISA) was analytically validated for diagnosis of pertussis, using the cut-off of 94 26

ELISA Units (EU)/mL. Little was known about the performance of this ELISA for 27

diagnosis in adults recently vaccinated with tetanus-diphtheria-acellular pertussis (Tdap), 28

which contains PT. The goal of this study was to determine when the assay can be used 29

following Tdap vaccination. 30

Methods. A cohort of 102 asymptomatic health care personnel (HCP) vaccinated with 31

Tdap (Adacel, Sanofi Pasteur) were aged 19 to 79 years (median 47 years) at vaccination. 32

For each HCP, specimens were available for evaluation at 2-10 time points (pre-33

vaccination to 24 months post-vaccination), and geometric mean concentrations (GMC) 34

for the cohort were calculated at each time point. Among 97 HCP who responded to 35

vaccination, a mixed model analysis with prediction and tolerance intervals was 36

performed to estimate the time at which serodiagnosis can be used following vaccination. 37

Results. The GMC was 8, 21, and 9 EU/mL at pre-vaccination, four, and 12 months post-38

vaccination, respectively. Eight of 102 (8%) HCP reached antibody titers ≥ 94 EU/mL 39

during their peak response but none had these titers by six months post-vaccination. 40

Calculated prediction and tolerance intervals were < 94 EU/mL by 45 and 75 days post-41

vaccination, respectively. 42

Conclusions. Tdap vaccination six months prior to testing did not confound result 43

interpretation. This seroassay remains a valuable diagnostic tool for adult pertussis. 44

45

INTRODUCTION 46


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


Pertussis, also known as whooping cough, is a highly contagious disease caused 47

by the bacterium Bordetella pertussis. Pertussis remains endemic in the United States 48

(U.S.) despite high vaccination coverage. Current diagnostic tools include real-time 49

polymerase chain reaction (r-PCR), culture, and serology. Patients, particularly 50

adolescents and adults, often do not present for diagnosis until later in the course of 51

infection, when culture and r-PCR assays are no longer as effective in detecting the 52

presence of the bacterium. Because two weeks are typically needed to mount an immune 53

response, serology can be a useful adjunct for later diagnosis. 54

Numerous pertussis serodiagnostic assays are currently available worldwide and 55

recommendations for testing, interpretation of results, and harmonization of assays have 56

been recently published (10, 23, 26). An IgG anti-PT ELISA was developed in 2004 by 57

the Center for Biologics Evaluation and Research (CBER) and the Centers for Disease 58

Control and Prevention (CDC) as a ready-to-use kit to diagnose pertussis infection in 59

adolescents and adults (19). In 2005, Tdap, the adult tetanus-diphtheria-acellular 60

pertussis vaccine that contains the same antigenic component as the ELISA, was licensed 61

and recommended for adolescents and adults (4, 16). Post-vaccination IgG anti-PT 62

antibody decay rates have been previously measured with various vaccine formulations, 63

study populations, and ELISA methods (5, 7, 17, 22). However, little is known about 64

whether Tdap booster vaccination will induce antibody levels that will confound 65

serodiagnostic interpretation. 66

In 2006, during a Tdap (Adacel, Sanofi Pasteur (SP)) vaccination campaign held 67

in response to a suspected pertussis outbreak, 106 asymptomatic healthcare personnel 68

(HCP) were enrolled in a study to measure (using an alternate ELISA) their booster 69


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


responses to vaccination. The initial protocol included pre-vaccination, and 1-, 2-, and 4-70

week(s) post-vaccination blood draws (15). We extended this study to include additional 71

blood samplings at 3, 6, 9, 12, 18, and 24 month post-vaccination so that the kinetics of 72

antibody response and decay could be further characterized specifically using the 73

serodiagnostic ELISA. The objective of this expanded study was to determine the 74

earliest time at which serodiagnosis can be used following Tdap vaccination. 75

76

MATERIALS/METHODS 77

Study protocol and ELISA Testing 78

Study protocol, including enrollment criteria, vaccine components, and 79

demographics of the 106 enrolled HCP, were previously described (15). Extension of the 80

study protocol was ethically approved by the Dartmouth College Committee for the 81

Protection of Human Subjects and the Institutional Review Board of the CDC. The 82

present study included additional samplings at 3, 6, 9, 12, 18, and 24 months post-83

vaccination. At the time of blood collection, HCP were asked about potential pertussis 84

illness or exposure. 85

Methods for the anti-PT IgG ELISA have been previously described (13, 19). 86

The diagnostic ELISA is a single point, single 1:100 dilution assay that contains six 87

ready-to-use standards and three ready-to-use controls (negative, intermediate, and 88

positive). The assay was originally calibrated to CBER Lot3 and later to the World 89

Health Organization (WHO) international reference standard 06/140 (29). All time 90

points corresponding to an enrollee were tested on the same ELISA plate and added in 91

duplicate wells. All values with a percent coefficient of variation (%CV) ≤ 25% were 92


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


considered valid. Reported values between 15-480 EU/mL, the range of the four 93

parameter logistic standard curve, were calculated as the average of two valid values 94

from separate plates run on two different days. Specimens with values < 15 EU/mL and 95

%CV > 25% were tested a maximum of three times; thus, reported values < 15 EU/mL 96

were either the average of two valid values or the average of three valid/invalid values. 97

Antibody concentrations were classified according to the proposed infection categories of 98

negative (< 49 EU/mL), indeterminate (49-93 EU/mL), and positive (≥ 94 EU/mL), and 99

according to other cut-off points of 125 and 200 EU/mL (2, 6, 18, 19). 100

101

Study Exclusions 102

Four HCP with baseline antibody level ≥ 49 EU/mL, indicative of possible active 103

infection by the proposed cut-off of 49 EU/mL (2, 19), were excluded, leaving 102 HCP 104

available for analysis (Figure 1). Of 101 HCP with known information on age at 105

vaccination, the median age was 47 years (range, 19 to 79 years), and of 100 HCP with 106

known information on sex, 59% were female. 107

Each of the 102 HCPs had antibody titers measured at 2 to 10 of the time points 108

(median, 7 measurements), and 31 subjects had antibody titers measured at all 10 time 109

points (Figure 1). Three HCPs had significant antibody increases (1.6 to 8.3-fold) that 110

occurred weeks to months after the initial booster response and may have indicated 111

possible exposure or infection; therefore, their increased antibody level measurements 112

and all subsequent measurements (n=10) were excluded. After these exclusions, a total 113

of 681 measurements from the 102 HCP were available for analysis. 114

115


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


Statistical Analyses 116

Analyses were based on log transformed antibody concentrations, log (antibody 117

concentration +1). Geometric mean concentrations (GMC) and their 95% confidence 118

intervals were calculated in two ways. First, GMCs using all values were calculated, 119

including those that fell outside the range of the standard curve. GMCs were also 120

calculated using a statistical model that censored values < 15 EU/mL and assumed all 121

values followed a normal distribution (12). The mean and standard deviation in the 122

model were estimated by maximum likelihood using the Newton-Raphson algorithm. 123

The Newton-Raphson algorithm was carried out using SAS PROC NLP (25). 124

A longitudinal mixed model analysis of antibody decay was used to estimate time 125

points at which certain antibody level thresholds were crossed (1, 9). Analysis for the 126

mixed modeling was restricted to HCP who had evidence of antibody response within the 127

first four weeks after vaccination and later decay. For each subject, we modeled the 128

antibody decay starting at the peak level. The mixed model was fit using restricted 129

maximum likelihood estimation in SAS PROC MIXED (24). The best-fitting model 130

included a random intercept and slope for each subject, and first order autoregressive 131

within subject covariance structure. 132

The population average antibody concentration for each day of the study period 133

was calculated based on the results of the mixed model. The 1-sided 95% upper 134

prediction limits that measure the uncertainty of the estimated average antibody 135

concentration for a single future specimen were then calculated. In addition to prediction 136

limits, 1-sided 95% upper tolerance limits that accounted for variation in the prediction 137

limits were also calculated by bootstrapping methods (8, 11). Tolerance intervals are 138


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


wider than prediction intervals because they contain at least a specified proportion (e.g., 139

95%) of the population with a high degree of confidence (e.g., 95%) (11, 14). 140

141

RESULTS 142

Four HCP experienced a prolonged cough and three HCP had been exposed to 143

someone with pertussis during the course of the extended study; however, only one HCP 144

was excluded because the baseline titer was > 49 EU/mL, indicative of possible recent 145

infection or exposure. Of the 94 HCP (Figure 1) who had all time points from baseline 146

through to four weeks post-vaccination, 66 (70%), 30 (32%), and 13 (14%) had a two, 147

four, and eight-fold increase in titer, respectively. Despite these increases, over half of all 148

specimens (397/681 or 58%) had antibody concentrations lower than the quantitative 149

range for the standard curve, i.e. < 15 EU/mL (Table 1). When values < 15 EU/mL were 150

modeled by a normal distribution, the GMCs changed only slightly at 8.2 EU/mL, 21.3 151

EU/mL, and 9.4 EU/mL at pre-vaccination, four weeks post-vaccination, and 12 months 152

post-vaccination, respectively (Table 1). 153

Similar antibody response patterns were observed among the HCP throughout the 154

study period, with the widest difference in concentration observed during the peak of 155

antibody response, at two and four weeks post-vaccination (Figure 2A). The typical 156

antibody decay profile was characterized by a rapid rise of titers within four weeks of 157

vaccination followed by an equally rapid fall of titers and then a slower decline or plateau 158

(Figure 2B). GMCs were classified by sampling times, with the lowest and highest 159

concentrations observed at 7.1 and 21.6 EU/mL for the pre-vaccination and four week 160

post-vaccination sampling times, respectively (Table 1). The GMC decreased to 16.3 161


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


EU/mL at three months post-vaccination and finally stabilized at 10.8 EU/mL at 12 162

months post-vaccination (Table 1). 163

Only 8 of 102 (8%) HCP ever had concentrations ≥ 94 EU/mL, and fewer still 164

(4% and 1%) reached the higher proposed cut-offs of 125 EU/mL and 200 EU/mL, 165

respectively (Table 2). Of the eight subjects whose titers exceeded the ELISA positive 166

cut-off, five discontinued enrollment after four weeks; however, titers for three of these 167

five subjects were stable or declining by their four week post-vaccination blood draw 168

(Figure 3). The remaining three subjects that continued in the study had values below the 169

proposed diagnostic cut-off of 94 EU/mL by six months post-vaccination. 170

A longitudinal mixed model analysis was employed to model the antibody profile 171

of a larger population vaccinated with Tdap (Figure 4). In our cohort of 102 HCP, the 172

number of study subjects with available specimens dropped 39% from 94 subjects at 4 173

weeks post-vaccination to 57 subjects at 3 months post-vaccination, with subsequent 174

attrition during the rest of the study period. The longitudinal analysis assumed that any 175

missing data were missing at random. Five of the 102 subjects were excluded because 176

they had very little (< 10%) to no increase in antibody level after vaccination during the 177

first four weeks after vaccination (n=4) or low antibody levels (< 4 EU/mL) throughout 178

the entire study (n=1), leaving 97 subjects for further analysis (Figures 1, 4). Accounting 179

for both the exclusion of 5 subjects and modeling each of the 97 antibody profiles from 180

the peak level, the number of measurements per subject ranged from one to nine (median 181

4), for a total of 407 measurements. The estimated population mean from the mixed 182

model fell in the middle of the individual profiles, indicating a good fit to the data (Figure 183

4). 184


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


To determine the earliest time at which serodiagnosis can be used following Tdap 185

vaccination, 1-sided 95% upper prediction and tolerance limits at each time point for 186

future specimens were used. The prediction limit for a single future specimen suggested 187

that the antibody level would be < 94 EU/mL by 45 days after vaccination (Figure 5). 188

The tolerance limit suggested that the predicted antibody level for at least 95% of the 189

vaccinated population would be < 94 EU/mL by 75 days after vaccination (Figure 5). 190

191

DISCUSSION 192

In a population of Tdap-vaccinated HCP who demonstrated expected booster 193

responses to vaccination within the first three months, only 8% had levels of antibody 194

high enough to interfere with the diagnostic interpretation of the IgG anti-PT ELISA. 195

Based on the upper prediction limit estimated by our model, the use of serology for 196

diagnostic purposes should be considered valid in patients who have not received Tdap 197

vaccination in the prior 45 days. Using the upper tolerance limit (75 days) it appears that 198

a slightly longer time frame would provide even more confidence regarding the use of 199

serodiagnosis among a population that will now be getting routinely vaccinated. 200

An elevated IgG anti-PT titer is currently the most sensitive and specific indicator 201

of recent pertussis infection (10, 27). Previous kinetic studies have shown that HCP 202

vaccinated with acellular vaccines can have IgG anti-PT antibody concentrations as high 203

as ~331 EU/mL (22). In response, many countries have adopted specific waiting periods, 204

up to a year, before suggesting the use of serodiagnosis in a vaccinated patient (10). 205

However, study objectives, criteria, vaccine formulations, and ELISA methodology can 206

be highly variable among previously published work resulting in a wide range of 207


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


observed decay rates and kinetic patterns (5, 7, 17, 22). Our modeling results suggest a 208

waiting period of three months post-vaccination in order to use our serodiagnostic 209

ELISA. 210

While the GMCs were far below the diagnostic cut-off for a recent infection, 211

antibody levels of eight individuals still surpassed the diagnostic cut-off. A possible 212

explanation is these asymptomatic individuals had a higher response due to previous or 213

recent exposure, which could be likely since the cohort is comprised of HCP. High 214

antibody titers have been observed in previous population studies and may simply be a 215

reflection of the normal variation observed in the population (2). 216

This study was an extension of a study which measured antibody levels one, two 217

and four weeks post-vaccination using a different ELISA performed at another laboratory 218

(15). IgG anti-PT GMCs were much higher in that study than those seen here (112 219

EU/mL rather than 19.0 EU/mL and 109.5 EU/mL rather than 21.3 EU/mL for the two 220

and four week post-vaccination sampling times, respectively). Further inspection led us 221

to examine if the PT antigen source alone or in combination with possible differences in 222

ELISA methodology could have been a potential source for the differences in 223

concentrations. Re-analysis of these sera by SP using a more highly purified antigen 224

preparation produced values lower than those originally reported (personal 225

communication with SP). These results led to a recently completed study to compare the 226

effect of four sources of PT antigen preparations on four different ELISA methods. 227

Results demonstrated that if the preparations are highly purified and well qualified and 228

the ELISAs are calibrated to a reference standard and well validated, these different PT 229

sources could be interchangeably used (13). 230


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


One important limitation of our study is that the data are derived from subjects 231

who received only one Tdap formulation. Vaccine formulations can differ by antigen 232

type, quantity, and purification or detoxification processes. This study used the Tdap 233

formulation, Adacel from SP (Lyon, France), which contains 2.5 µg of PT. Formulations 234

that contain higher amounts of PT may elicit a greater antibody response that could take 235

longer to decay and thus delay the time frame for the applicability of serodiagnosis with 236

the ELISA. Recent vaccine response studies with a formulation that contains a higher 237

concentration of PT (8 μg) observed higher peak post-vaccination GMCs (126.5, 90.3, 238

and 62.7 EL.U/mL) (3, 20, 28); one observed a decay sampling at one year post-239

vaccination with a GMC of 22.7 EL.U/mL (28). Based on the data of these studies and 240

the well-recognized rapid decline in titers soon after the peak, we believe that a six-241

month waiting period after vaccination is sufficient. 242

Another potential limitation is that post-vaccination titers in adolescents may be 243

different from adults. The time between vaccinations for adolescents is much shorter 244

than adults and therefore may elicit a higher response to the booster. However, Rieber, et 245

al. observed post-Tdap booster GMCs of 16.4, 17.1 and 50.3 EU/mL one month after 246

vaccination for adolescents with a 5-dose acellular, a 4-dose acellular, or a 4-dose whole-247

cell vaccine primary series, respectively (21). In addition, Mertsola, et al. observed 248

comparable antibody decay profiles from booster vaccinations in adolescents and adults 249

(20). These findings suggest that our results for adults with the diagnostic ELISA may be 250

similarly interpreted for adolescents. 251

The need for a serological diagnostic tool has been increasing substantially as the 252

public health community seeks to understand the true burden of pertussis. The IgG anti-253


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


PT assay is an ideal complement to culture and PCR to determine infection in the later 254

phases of disease and in adolescents and adults who do not display typical pertussis 255

symptoms. The late nature of reporting is the primary reason that outbreak investigations 256

are often retrospective and dependent on serological testing for confirmation. Given 257

recent initiatives to improve Tdap vaccination in adolescents and adults, clarifying 258

confounding interpretations of serological results is crucial. 259

In summary, our study predicts that the IgG anti-PT ELISA will remain useful to 260

measure PT antibody levels in Tdap-vaccinated patients beginning as early as 75 days 261

post vaccination. Certainly by six months post-vaccination, predicted antibody levels of 262

vaccinated adult populations fell far below the diagnostic cut-off, making interference 263

with serodiagnosis interpretation quite unlikely. For epidemiological studies and as part 264

of a public health response, public health laboratories may use this ELISA for pertussis 265

diagnostics regardless of vaccination history. Clinicians managing individual patients 266

should maintain a more comprehensive approach to diagnosing pertussis in a recently 267

vaccinated person, considering symptoms and the appropriateness of other diagnostic 268

tools, but should feel confident using this serologic test if vaccination has occurred more 269

than six months previously. 270

271

ACKNOWLEDGEMENTS 272

We would like to thank GlaxoSmithKline for kindly providing the pertussis toxin and the 273

Center for Biologics Evaluation and Research for the CBER Lot3 reference sera. We are 274

also grateful to Terre Currie for her work with recruitment and collection of specimens. 275

276


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


Disclaimer: The findings and conclusions in this report are those of the author(s) of 277

each section and do not necessarily represent the entire group of participants or the 278

official position of the Centers for Disease Control and Prevention/Agency for Toxic 279

Substances and Disease Registry. 280

281

REFERENCES 282

1. Amanna, I. J., N. E. Carlson, and M. K. Slifka. 2007. Duration of humoral 283

immunity to common viral and vaccine antigens. N Engl J Med 357:1903-15. 284

2. Baughman, A. L., K. M. Bisgard, K. M. Edwards, D. Guris, M. D. Decker, K. 285

Holland, B. D. Meade, and F. Lynn. 2004. Establishment of diagnostic cutoff 286

points for levels of serum antibodies to pertussis toxin, filamentous 287

hemagglutinin, and fimbriae in adolescents and adults in the United States. Clin 288

Diagn Lab Immunol 11:1045-53. 289

3. Booy, R., O. Van der Meeren, S. P. Ng, F. Celzo, G. Ramakrishnan, and J. 290

M. Jacquet. 2010. A decennial booster dose of reduced antigen content 291

diphtheria, tetanus, acellular pertussis vaccine (Boostrix) is immunogenic and 292

well tolerated in adults. Vaccine 29:45-50. 293

4. Broder, K. R., M. M. Cortese, J. K. Iskander, K. Kretsinger, B. A. Slade, K. 294

H. Brown, C. M. Mijalski, T. Tiwari, E. J. Weston, A. C. Cohn, P. U. 295

Srivastava, J. S. Moran, B. Schwartz, and T. V. Murphy. 2006. Preventing 296

tetanus, diphtheria, and pertussis among adolescents: use of tetanus toxoid, 297

reduced diphtheria toxoid and acellular pertussis vaccines recommendations of 298


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


the Advisory Committee on Immunization Practices (ACIP). MMWR Recomm 299

Rep 55:1-34. 300

5. Dalby, T., J. W. Petersen, Z. B. Harboe, and K. A. Krogfelt. 2010. Antibody 301

responses to pertussis toxin display different kinetics after clinical Bordetella 302

pertussis infection than after vaccination with an acellular pertussis vaccine. J 303

Med Microbiol 59:1029-36. 304

6. de Melker, H. E., F. G. Versteegh, M. A. Conyn-Van Spaendonck, L. H. 305

Elvers, G. A. Berbers, A. van Der Zee, and J. F. Schellekens. 2000. Specificity 306

and sensitivity of high levels of immunoglobulin G antibodies against pertussis 307

toxin in a single serum sample for diagnosis of infection with Bordetella pertussis. 308

J Clin Microbiol 38:800-6. 309

7. Edelman, K., Q. He, J. Makinen, A. Sahlberg, M. Haanpera, L. Schuerman, 310

J. Wolter, and J. Mertsola. 2007. Immunity to pertussis 5 years after booster 311

immunization during adolescence. Clin Infect Dis 44:1271-7. 312

8. Efron, B., and R. J. Tibshirani. 1998. An Introduction to the Bootstrap. 313

Chapman & Hall/CRC, NY. 314

9. Fraser, C., J. E. Tomassini, L. Xi, G. Golm, M. Watson, A. R. Giuliano, E. 315

Barr, and K. A. Ault. 2007. Modeling the long-term antibody response of a 316

human papillomavirus (HPV) virus-like particle (VLP) type 16 prophylactic 317

vaccine. Vaccine 25:4324-33. 318

10. Guiso, N., G. Berbers, N. K. Fry, Q. He, M. Riffelmann, and C. H. Wirsing 319

von Konig. 2011. What to do and what not to do in serological diagnosis of 320


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


pertussis: recommendations from EU reference laboratories. Eur J Clin Microbiol 321

Infect Dis 30:307-12. 322

11. Hahn, G., and W. Q. Meeker. 1991. Statistical intervals: a guide for 323

practitioners. John Wiley & Sons, Inc., NY. 324

12. Hornung, R. W., and L. D. Reed. 1990. Estimation of Average Concentration in 325

the Presence of Nondetectable Values. Applied Occupational and Environmental 326

Hygiene 5:46-51. 327

13. Kapasi, A., B. D. Meade, B. Plikaytis, L. Pawloski, M. D. Martin, S. Yoder, 328

M. T. Rock, S. Coddens, V. Haezebroeck, F. Fievet-Groyne, G. Bixler, C. 329

Jones, S. Hildreth, K. M. Edwards, N. E. Messonnier, and M. L. Tondella. 330

2012. Comparative Study of Different Sources of Pertussis Toxin (PT) as Coating 331

Antigens in IgG Anti-PT Enzyme-Linked Immunosorbent Assays. Clin Vaccine 332

Immunol 19:64-72. 333

14. Katki, H. A., E. A. Engels, and P. S. Rosenberg. 2005. Assessing uncertainty in 334

reference intervals via tolerance intervals: application to a mixed model 335

describing HIV infection. Stat Med 24:3185-98. 336

15. Kirkland, K. B., E. A. Talbot, M. D. Decker, and K. M. Edwards. 2009. 337

Kinetics of pertussis immune responses to tetanus-diphtheria-acellular pertussis 338

vaccine in health care personnel: implications for outbreak control. Clin Infect Dis 339

49:584-7. 340

16. Kretsinger, K., K. R. Broder, M. M. Cortese, M. P. Joyce, I. Ortega-Sanchez, 341

G. M. Lee, T. Tiwari, A. C. Cohn, B. A. Slade, J. K. Iskander, C. M. Mijalski, 342

K. H. Brown, and T. V. Murphy. 2006. Preventing tetanus, diphtheria, and 343


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


pertussis among adults: use of tetanus toxoid, reduced diphtheria toxoid and 344

acellular pertussis vaccine recommendations of the Advisory Committee on 345

Immunization Practices (ACIP) and recommendation of ACIP, supported by the 346

Healthcare Infection Control Practices Advisory Committee (HICPAC), for use of 347

Tdap among health-care personnel. MMWR Recomm Rep 55:1-37. 348

17. Le, T., J. D. Cherry, S. J. Chang, M. D. Knoll, M. L. Lee, S. Barenkamp, D. 349

Bernstein, R. Edelman, K. M. Edwards, D. Greenberg, W. Keitel, J. Treanor, 350

and J. I. Ward. 2004. Immune responses and antibody decay after immunization 351

of adolescents and adults with an acellular pertussis vaccine: the APERT Study. J 352

Infect Dis 190:535-44. 353

18. Marchant, C. D., A. M. Loughlin, S. M. Lett, C. W. Todd, L. H. Wetterlow, 354

R. Bicchieri, S. Higham, P. Etkind, E. Silva, and G. R. Siber. 1994. Pertussis 355

in Massachusetts, 1981-1991: incidence, serologic diagnosis, and vaccine 356

effectiveness. J Infect Dis 169:1297-305. 357

19. Menzies, S. L., V. Kadwad, L. C. Pawloski, T. L. Lin, A. L. Baughman, M. 358

Martin, M. L. Tondella, and B. D. Meade. 2009. Development and analytical 359

validation of an immunoassay for quantifying serum anti-pertussis toxin 360

antibodies resulting from Bordetella pertussis infection. Clin Vaccine Immunol 361

16:1781-8. 362

20. Mertsola, J., O. Van Der Meeren, Q. He, A. Linko-Parvinen, G. 363

Ramakrishnan, L. Mannermaa, M. Soila, M. Pulkkinen, and J. M. Jacquet. 364

2010. Decennial administration of a reduced antigen content diphtheria and 365


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


tetanus toxoids and acellular pertussis vaccine in young adults. Clin Infect Dis 366

51:656-62. 367

21. Rieber, N., A. Graf, B. H. Belohradsky, D. Hartl, S. Urschel, M. Riffelmann, 368

C. H. Wirsing von Konig, and J. Liese. 2008. Differences of humoral and 369

cellular immune response to an acellular pertussis booster in adolescents with a 370

whole cell or acellular primary vaccination. Vaccine 26:6929-35. 371

22. Riffelmann, M., M. Littmann, C. Hulsse, and C. H. von Konig. 2009. 372

Antibody decay after immunisation of health-care workers with an acellular 373

pertussis vaccine. Eur J Clin Microbiol Infect Dis 28:275-9. 374

23. Riffelmann, M., K. Thiel, J. Schmetz, and C. H. Wirsing von Koenig. 2010. 375

Performance of commercial enzyme-linked immunosorbent assays for detection 376

of antibodies to Bordetella pertussis. J Clin Microbiol 48:4459-63. 377

24. SAS Institute, Inc. 2009. The Mixed Procedure. SAS/STAT 9.2 User's Guide, 378

Second ed. SAS Institute, Inc., Cary, NC. 379

25. SAS Institute, Inc. 2010. The NLP Procedure. SAS/OR 9.22 User's Guide: 380

Mathematical Programming. SAS Institute, Inc., Cary, NC. 381

26. Tondella, M. L., G. M. Carlone, N. Messonnier, C. P. Quinn, B. D. Meade, D. 382

L. Burns, J. D. Cherry, N. Guiso, E. L. Hewlett, K. M. Edwards, D. Xing, A. 383

Giammanco, C. H. Wirsing von Konig, L. Han, L. Hueston, J. B. Robbins, M. 384

Powell, C. M. Mink, J. T. Poolman, S. W. Hildreth, F. Lynn, and A. Morris. 385

2009. International Bordetella pertussis assay standardization and harmonization 386

meeting report. Centers for Disease Control and Prevention, Atlanta, Georgia, 387

United States, 19-20 July 2007. Vaccine 27:803-14. 388


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


27. Watanabe, M., B. Connelly, and A. A. Weiss. 2006. Characterization of 389

serological responses to pertussis. Clin Vaccine Immunol 13:341-8. 390

28. Weston, W., M. Messier, L. R. Friedland, X. Wu, and B. Howe. 2011. 391

Persistence of antibodies 3 years after booster vaccination of adults with 392

combined acellular pertussis, diphtheria and tetanus toxoids vaccine. Vaccine 393

29:8483-6. 394

29. Xing, D., C. H. Wirsing von Konig, P. Newland, M. Riffelmann, B. D. Meade, 395

M. Corbel, and R. Gaines-Das. 2009. Characterization of reference materials for 396

human antiserum to pertussis antigens by an international collaborative study. 397

Clin Vaccine Immunol 16:303-11. 398

399

FIGURE LEGENDS 400

FIGURE 1. Flow chart of study populations for analysis. HCP = health care personnel; 401

GMC = geometric mean concentration 402

403

FIGURE 2. (A) Box plot of antibody concentrations in 102 vaccinated subjects, by 404

sampling time. Each box displays the 25th percentile, median, mean (+), and 75th 405

percentile. Whiskers are 1.5 times the interquartile range in length. Outliers are 406

displayed as stars. Note that the x-axis is not drawn to scale. The dashed line represents 407

the proposed cut-off of 94 EU/mL. (B) Geometric mean antibody concentrations (solid 408

line) in 102 vaccinated subjects and 95% confidence intervals in Tdap-vaccinated 409

subjects, by sampling time. The dashed line represents the proposed cut-off of 94 410

EU/mL. 411

412


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


FIGURE 3. Antibody peak and decline patterns for the eight subjects that surpassed the 413

94 EU/mL cut-off (dashed line). 414

415

FIGURE 4. Time plot of antibody level versus time after vaccination (in days) for 97 416

subjects, beginning from the peak antibody level. The estimated population mean (thick 417

line) from the mixed model is superimposed on the plot. 418

419

FIGURE 5. Plot of estimated population mean (thick line) after the peak antibody 420

response, based on the mixed model analysis for 97 subjects with the pointwise 1-sided 421

95% upper prediction limit (solid line) and the pointwise 1-sided 95% upper tolerance 422

limit (dashed line). Reference lines for the proposed thresholds at 94 EU/ml, 125 423

EU/mL, and 200 EU/mL are superimposed on the plot (gray dashed lines). 424


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

FIGURE 1.

4 HCP excluded because baseline titer was ≥ 49

/

106 HCP originally enrolled

102 HCP with 2-10 time points (691 measurements)

EU/mL

10 measurements from 3 HCP excluded (1.6 to 8.3-fold antibody increase from previous time point, after peak response)

102 HCP analyzed for GMCs and positivity

(681 measurements)

97 HCP analyzed from antibody

5 HCP excluded (< 10% antibody response)

97 HCP analyzed from antibody peak for longitudinal model

(407 measurements)


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

FIGURE 2.

A.

B. on February 2, 2020 by guest

http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

FIGURE 3FIGURE 3.


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

FIGURE 4.


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

FIGURE 5.


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


TABLE 1. Geometric mean concentration (GMC) (EU/mL) of IgG anti-PT levels in 102

vaccinated subjects by sampling time. w = week(s) post-vaccination; m = months post-

vaccination

Sampling Time n† GMC (95% CI)

All Values Values < 15 EU/mL

(%) GMC (95% CI)

Model*

Baseline 102 7.1 (6.0–8.4) 82 (80.4) 8.2 (7.1–9.4)

1 week (0.9–2.4 w) 98 11.4 (9.4–13.7) 62 (63.3) 10.7 (8.8–13.0)

2 weeks (1.9–4.0 w) 96 20.3 (16.7–24.5) 40 (41.7) 19.0 (15.6–23.3)

4 weeks (3.6–6.9 w) 94 21.6 (18.0–25.9) 33 (35.1) 21.3 (17.7–25.6)

3 months (1.1–4.2 m) 57 16.3 (12.8–20.8) 26 (45.6) 16.1 (12.7–20.4)

6 months (5.7–7.2 m) 54 13.3 (10.4–16.9) 31 (57.4) 12.8 (10.1–16.2)

9 months (9.0–12.4 m) 47 12.0 (9.4–15.3) 30 (63.8) 11.0 (8.6–14.1)

12 months (11.8–13.6 m) 51 10.8 (8.6–13.5) 36 (70.6) 9.4 (7.4–12.0)

18 months (17.8–19.1 m) 43 11.1 (8.6–14.1) 30 (69.8) 9.8 (7.5–12.6)

24 months (23.9–25.0 m) 39 10.0 (7.6–13.1) 27 (69.2) 10.1 (7.8–13.0) *GMC and 95% CI were estimated from a statistical model that censored values < 15 EU/mL and assumed

all values followed a normal distribution.

†The number of measurements available at each sampling time for the entire cohort. Note that for several

subjects, specimens were not available for all sampling times between their pre-vaccination and last

sampling times.


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/


TABLE 2. Number of positive specimens (%) by various proposed cut-offs by sampling

time: 94 EU/mL, 125 EU/mL, and 200 EU/mL. n = sample size; w = week(s) post-

vaccination; m = months post-vaccination

†The number of measurements available at each sampling time for the entire cohort. Note that for several

subjects, specimens were not available for all sampling times between their pre-vaccination and last

sampling times.

Sampling Time n† Proposed Cut-offs

94 EU/mL 125 EU/mL 200 EU/mL

Baseline 102 0 (0) 0 (0) 0 (0)

1 w 98 2 (2) 0 (0) 0 (0)

2 w 96 6 (6.2) 2 (2.1) 1 (1)

4 w 94 6 (6.4) 3 (3.2) 0 (0)

3 m 57 2 (3.5) 1 (1.8) 0 (0)

6 m 54 0 (0) 0 (0) 0 (0)

9 m 47 0 (0) 0 (0) 0 (0)

12 m 51 0 (0) 0 (0) 0 (0)

18 m 43 0 (0) 0 (0) 0 (0)

24 m 39 0 (0) 0 (0) 0 (0)


http://cvi.asm.org/

Dow

nloaded from

http://cvi.asm.org/

Documents

on January 5, 2020 by guest · 60 adolescents and adults (19). In 2005, Td ap, the adult tetanu s-diphtheria-acellular 61 pertussis vaccine that contai ns the same antigenic compone