Routine immunization with diphtheria, tetanus and whole cell pertussis (wP) vaccine began in Israel in the late 1950s and led to a marked decrease in the national incidence of pertussis disease. Acellular formulations (acellular pertussis, aP) were introduced in 2002 with wide acceptance; yet the incidence rate of pertussis still increased in 2003–2004 prompting additional diphtheria–tetanus–aP (dTaP) booster doses to the national immunization policy. Israel’s current pertussis immunization policy includes 2 aP formulations, Infanrix, GlaxoSmithKline (Rixensart, Belgium) or Poliacel, Sanofi Pasteur (Toronto, Canada), administered at ages 2, 4, 6 and 12 months and dTaP-inactivated polio virus (dTap-IPV, Boostrix Polio, GlaxoSmithKline) for 7-year to 8-year-old children and dTap (Boostrix, GlaxoSmithKline) dose for 13-year to 14-year-old children (Fig. 1).
Despite the high national vaccination coverage (95%) and the aforementioned boosters, there has been a resurgence of pertussis with estimated incidence rates peaking at 35 of 100,000 in 2007 and 2012 (Fig. 1).
The aim of our study was to examine different contributing factors that might have led to the resurgence of pertussis in Israel despite the addition of dTap booster doses. We sought to examine the laboratory detection rates of B. pertussis at Bnai Zion’s pertussis reference laboratory, 2 years after the addition of the pertussis last adolescent booster dose. Moreover, we sought to analyze representative circulating isolates of pertussis by pulsed-field gel electrophoresis (PFGE) and to identify pertussis isolates not expressing pertactin (Prn) protein.
MATERIALS AND METHODS
Real-time Polymerase Chain Reaction and Culture
Real time polymerase chain reaction (PCR) and culture data were examined, January 1, 2010 to December 31, 2013. For the entire study period, the same laboratory techniques were utilized. For PCR, B. pertussis detection was based on the identification of insertion sequence 481 and the absence of insertion sequence 1001 as previously described.1
For culture, samples were plated on Regan-Lowe Bordetella agar and incubated at 37°C for 14 days as previously described.1
Pulsed-field Gel Electrophoresis
PFGE was performed in the Clinical Microbiology Laboratory at Rambam Medical Center, Haifa, Israel, as previously described by Mooi et al2 utilizing the SpeI restriction enzyme.
Immunoblot Analysis of Pertactin
The Prn protein was detected by Western immunoblot. Bacterial suspensions of 0.6 optical density (600 nm) were centrifuged, mixed with Laemmli loading buffer (4×, +5% beta- mercaptoethanol) and boiled for 10 minutes at 100°C.
Proteins were separated on sodium dodecyl sulfate–polyacrylamide 14% gel electrophoresis at 120 V for 1.5 hours and transferred to nitrocellulose membrane at 200 mA for 2 hours. Membranes were blocked on ice with 5% milk for 1 hour and incubated overnight with polyclonal antibody against Prn (made in sheep, The National Institute for Biological Standards and Control cat# 97/558) diluted 1:5000. Secondary antibody donkey polyclonal to sheep IgG Heavy and Light—horse radish peroxidase (cat# Abcam AB-ab6900) was used at 1:10,000 dilution in Tris-buffered saline with tween for 1 hour. Enhanced chemiluminescence reagent (Pierce) was added following manufacturer recommendations. And films were exposed for 1–3 minutes and developed with Fujifilm reagents.
Bands of 69 kDa were detected and reported as positive or negative for each sample.
The χ2 test for trends was used to test differences in detection rate of positive pertussis PCR and culture positive specimens. Bonferroni corrections were applied to ensure a confidence level of 95% for pairwise comparisons between the years. Analysis was performed via the Windows Program for Epidemiologists.
Pertussis Laboratory Detection by PCR and Culture
We found a statistically significant change in the B. pertussis PCR positivity rate, 40 of 408 (9.8%), 66 of 491 (13.4%), 111 of 504 (22%) and 87 of 572 (15.2%) for the years 2010, 2011, 2012 and 2013, P < 0.001, respectively. Post-hoc testing revealed that in 2012, there was a significantly higher PCR positivity rate than 2010, 2011 and 2013, P < 0.001, P < 0.002 and P < 0.02, respectively.
A similar trend was also evident for culture-positive specimens, 19 of 362 (5.2%), 29 of 356 (8.14%), 45 of 416 (10.8%) and 27 of 572 (4.7%) for 2010, 2011, 2012 and 2013, P < 0.001, respectively. Post-hoc testing revealed that during 2012 there was a significantly higher culture detection rate than 2010 and 2013, P < 0.02 and P < 0.001, respectively.
Pulsed-field Gel Electrophoresis Analysis
Four PFGE restriction profiles were isolated, referenced to the 2007–2008 restriction patterns named A, B, C and D.3
Subsequent select analysis during the years 2009–2012 revealed PFGE profile A in 10 of 38 samples (26%), B in 19 of 38 (50%) and the disappearance of profiles C and D.
The decrease in the frequency of PFGE profile A during 2009–2012, in comparison with 2007–2008, 54% (44 of 82) versus 26% (10 of 38) was statistically significant, P < 0.006. Moreover, new distinct strains emerged and were named E (2 of 38, 5%), F (1 of 38, 3%), G (3 of 38, 8%), H (1 of 38, 3%) and I (2 of 38, 5%).
Identification of B. pertussis not Expressing Pertactin
During the study period, 65 isolates were assessed for the expression of Prn. The first isolate not expressing Prn was identified in an isolate from the year 2006. However, the proportion of Prn-deficient isolates increased significantly over the years, 2005–2006, 2011–2012 and 2013–2014, 6.6% (1 of 15) versus 7.1% (1 of 17) versus 33.3% (11 of 33), P < 0.03.
Our study showed that despite the addition of 2 dTaP booster doses to Israel’s national immunization policy, the national pertussis incidence rate remained high. There are multiple factors believed to contribute to this high burden of disease. It is well established that cyclic peaks of pertussis occur at 2–5 year intervals in the post vaccination era.4 Moreover, waning immunity and decreased efficacy of the aP vaccine in comparison with the whole cell composition have also been reported.5,6 Sheridan et al7 inferred that the ongoing pertussis epidemic in Australia might be attributed to the fact that children are not primed with any wP vaccine. Additionally, Tartof et al6 found a steady increase in the risk of pertussis after a 5-dose aP series likely attributed to its waning immunity. It should be emphasized, however, that despite the aforementioned concern regarding immunization with aP, the World Health Organization’s most recent report stated that both aP and wP vaccines are efficacious in preventing pertussis disease.8
Regardless of the pertussis vaccine type utilized, high vaccination coverage may impose vaccine pressure thereby leading to pertussis antigenic drift.5 Indeed, our group previously reported on the predominance of 2 closely related PFGE profiles in Israel, during a resurgence of pertussis, 2007–2008 with the most common PFGE profile A, having the same PFGE cluster of the dominant European BpSR11 strain (PFGE cluster IVβ) and the second common PFGE profile B was grouped to PFGE cluster IVα.3 Moreover, during the subsequent 4 years, we found a marked decrease in the frequency of PFGE profile A (PFGE cluster IVβ), an increase in PFGE profile B (PFGE cluster IVα) and identified new patterns.
B. pertussis circulating isolates with a similar PFGE clusters were also observed in Europe since 1998.9 Specifically, BpSR11 (PFGE cluster IVβ) and BpSR10 strains (PFGE cluster IVα) corresponded to profiles A and B, respectively.9
In addition to the changes in the PFGE, we observed a dramatic increase in the proportion of B. pertussis isolates not expressing Prn, associated with the addition of booster aP vaccines, reaching 33% in the years 2013–2014. Prn is a virulence factor of the B. pertussis bacterium and enhances the adherence of B. pertussis to ciliated respiratory epithelium and is a constituent of most aP vaccine formulations.10 It should be noted that the proportion of Prn-deficient isolates might be affected by the vaccination strategy and coverage in different countries.11–14 In Japan, Prn-deficient isolates were detected from isolates as early as 1995–1999.11 Most recently, an Australian study reported its countries highest proportion of Prn-deficient isolates.12
Lastly, enhanced awareness of the disease among healthcare providers may have prompted increased sampling with sensitive laboratory diagnostics, for example, PCR, which might have contributed to the B. pertussis high incidence rates. As Kaczmarek et al15 reported, there was a 7-fold rise in the likelihood of pertussis test requests, in a stable set of Australian general practice encounters, in 2000–2011 demonstrating a changing trend in pertussis testing. We demonstrated that the rate of positivity of PCR changed also over time along with the dynamic changes in the national incidence rate of pertussis.
Despite the small sample size, these findings demonstrate the importance of continued surveillance of the genotypic and phenotypic characteristics of B. pertussis circulating isolates and continued effort to develop a more durable preventative pertussis vaccine.
The authors thank Michael Peterman, MSc and Tanya Gorvetsh of the Clinical Microbiology Unit at Bnai Zion Medical Center, Haifa, Israel, for performing the pertussis PCR testing and assisting in the PFGE analysis. The authors also greatly acknowledge Professor Nicole Guiso of the Louis Pasteur Institute and dedicated laboratory team for mentoring our lab with their Immunoblot technique for pertactin analysis.
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