Despite the presence of universal immunization programs and high vaccination coverage, pertussis remains a major public health concern. Pertussis epidemics occur every 3–4 years because infections and the vaccines do not induce lifelong immunity to the disease.1,2 In Italy, the disease peaks in spring and summer, namely, between March and August.3 In infants, pertussis is an important cause of death: case-fatality rates range from 0.2% in developed countries to 4% in developing countries.4 The severity of the symptoms correlates inversely with not only the age of the patient but also their immunization status: unimmunized children present with the typical clinical features and complications,5,6 whereas immunized children, adults and adolescents usually have a milder course with atypical clinical features.7,8 While the atypical clinical forms of pertussis in partially immune children or adolescents and adults can be difficult to recognize, the absence of typical symptoms does not exclude the diagnosis of pertussis.9,10 Indeed, in 2012, the European Centre for Disease Prevention and Control added apnoeic episodes to the clinical diagnostic criteria to increase the sensitivity with which pertussis is diagnosed in infants who lack other clinical manifestations.11 It can also be difficult to diagnose pertussis during the winter season because of the circulation of other pathogens, such as respiratory syncytial virus.9 The protection generated by both natural and acquired immunization is believed to wane after 4–12 years.12 Parents and household members are often the primary source of pertussis transmission for newborn babies and unvaccinated and incompletely immunized children.13 In Italy, the acellular vaccine is administered via the hexavalent combined vaccine during the first year of life with a primary 3-dose schedule (3rd, 5th and 11th month). Booster doses at 5–6 years and 11–12 years of age are recommended to avoid infections due to the waning of immunity.14,15
Despite high vaccination coverage among children, several countries have experienced a resurgence of pertussis starting in the 1990s.16 In Europe, although the number of pertussis cases reported to World Health Organization tended to fluctuate between 2000 and 2013, there was a marked increase in 201217: Austria, the Czech Republic, Germany, Poland, Spain, Denmark and Switzerland all reported an increase in pertussis case numbers at this time point.18 Moreover, between 2000 and 2013, the Netherlands and Norway reported the highest number of pertussis cases. Indeed, between 2007 and 2013, these 2 countries had 47% of the total number of pertussis reported cases in the European region.18 In Italy, a study that was based on routine surveillance system data did not report any increases in pertussis notification rates between 2000 and 2009.3 However, another Italian study showed that, between 1999 and 2009, there was an increase in infant (<1 year of age) hospitalization rates due to pertussis.15
The resurgence of pertussis worldwide has been attributed to several factors, including improved awareness of the disease, introduction of the acellular vaccine, genetic changes in Bordetella pertussis (BP) and the gradual loss of protective immunity after natural infection and vaccination.19,20 Of particular concern are the genetic changes in several surface-associated BP proteins that elicit protective immunity, namely, pertussis toxin (ptx), pertactin (prn) and fimbriae.21,22 In recent years, the predominant BP strain has changed from one containing the ptxP1 allele (which controls the synthesis and export of ptx) to a strain bearing the ptxP3 allele, which increases ptx production.16 Moreover, BP strains that do not express prn have been isolated; they are named prn-deficient strains. However, at present, it seems that prn-deficient BP strains do not differ from the prn-producing strains in terms of the clinical disease they cause.23
The aim of the present study was to describe the epidemiology of pertussis in the Puglia region in 2006–2015. The relevant epidemiologic data were extracted from several different data sources. In addition, 80 BP-positive samples that were collected between 2010 and 2016 were subjected to molecular typing to determine their genetic profiles.
Epidemiologic Data Sources
The numbers of pertussis cases in children (<18 years of age) that were recorded in 2006–2015 were extracted from the National Hospital Discharge Database and the Information System of Infectious Diseases (termed Sistema Informativo delle Malattie Infettive in Italian). Moreover, the numbers of pertussis diagnoses in children that were confirmed in 2014–2015 by the Regional Reference Laboratory (RRL) of Puglia were extracted from the RRL database. To avoid duplicates, the cases detected in the 3 data sets were matched by name, date of birth and sex.
The National Hospital Discharge Database contains the hospital discharge forms (termed Scheda Dimissione Ospedaliera in Italian) that are filled out for all patients who are discharged from any public or private hospital in Italy.15 The Scheda Dimissione Ospedalieras are coded according to the International Classification of Diseases, 9th revision, Clinical Modification.24 The Scheda Dimissione Ospedalieras from the Puglia region database that were filled out between 2006 and 2015 and contained the following International Classification of Diseases, 9th revision, Clinical Modification codes were included in our analysis: 033.0 (pertussis due to BP), 033.1 (pertussis due to Bordetella parapertussis), 033.8 (pertussis due to other specified pathogens), 033.9 (pertussis due to unspecified pathogens) and 484.3 (pneumonia in pertussis).
Sistema Informativo delle Malattie Infettive is the routine surveillance system for infectious diseases in Italy.25 It collects all notifications of infectious diseases, as specified by the Italian Ministerial Decree of December 15, 1990.26 In Italy, local health authorities must be notified of all cases of pertussis within 48 hours. However, this passive surveillance system is affected by limitations, such as undernotification. Consequently, the pertussis data in this system are incomplete.15
The RRL that confirms the pertussis diagnoses in Puglia is the Laboratory of Molecular Epidemiology of the Hygiene Unit of Policlinico Hospital, Bari. Starting in April 2014, all hospitalized and nonhospitalized suspected cases of pertussis in the Puglia region were confirmed by polymerase chain reaction at this RRL.27 This RRL has recently undertaken a molecular analysis of the cases in Puglia that it confirmed between June 2014 and August 2016 to determine whether virulence-associated BP variants are emerging.
The vaccination status of the cases that were confirmed by the RRL was determined by consulting the Regional Immunization Registry. The vaccination status is categorized as fully vaccinated (≥3 doses), undervaccinated (<3 doses), unvaccinated (no doses), undervaccinated because the child was not old enough to receive all 3 doses (<3 doses) and unvaccinated because the child was too young to receive the first dose (no doses).
The vaccination coverage for pertussis was obtained from the routine detection system of the Regional Epidemiological Observatory. Notably, in Italy, regional or local service providers are responsible for vaccine procurement in Italy. Therefore, different hexavalent vaccine products may have been used during the study period. Such information has not been taken into consideration in the present analysis.
This study was conducted according to the tenets of the Declaration of Helsinki. Ethical approval was obtained from the Ethical Committee of Azienda Ospedaliero-Universitaria Policlinico of Bari. Informed written consent was obtained from the individuals who provided specimens: consent was obtained before the sample was obtained. In the case of children, written informed consent to provide a sample was obtained from the parents or the legal guardians.
Statistical Analysis of the Epidemiologic Data
All statistical analyses were performed using the software package STATA 11.0. Student t test was used to compare means while the χ2 test was used to compare proportions. Differences between means or proportions were considered to be statistically significant when the P value was below 0.05. Multivariate logistic regression analysis was performed to determine which demographic and clinical variables associated independently with hospitalization. The results were expressed as odds ratios and 95% confidence intervals.
Molecular Testing of Clinical Pertussis Samples
Since April 2014, the RRL in Bari has been collecting the pharyngeal swabs from all hospitalized and nonhospitalized children in whom there is a clinical suspicion of pertussis. The swabs are subjected to nucleic acid extraction using the MagNA Pure (Roche Diagnostics, Mannheim Germany). BP is identified using a commercial RealTime-polymerase chain reaction method (Anyplex RB5Detection Seegene Inc., Seoul, Republic of Korea).
Between June 2014 and August 2016, 83 BP-positive samples were collected by the RRL. In the present study, the DNAs of 70 of these clinical samples were subjected to molecular characterization (the remaining 13 samples were not available). Ten BP-positive clinical samples that were obtained during an outbreak of pertussis in Puglia in 20101 were also subjected to molecular characterization. Molecular characterization of BP was performed by sequencing the ptxA, ptxP and prn genes according to the method described previously.22,28 Moreover, 10 of the 70 BP-positive samples that were obtained between June 2014 and August 2016 were randomly selected and subjected to multiple-locus variable-number tandem repeat analysis (MLVA): all of these samples were obtained between 2015 and 2016. Six loci were sequenced in the MLVA as described previously.28,29 The MLVA type was assigned on the basis of the number of repeats in loci 1, 3a, 3b, 4, 5 and 6 using the International Reference Database (http://www.mlva.net/).
Incidence of Pertussis, Pertussis Vaccination Rates and Hospitalization Rates in 2006–2015
Between January 2006 and December 2015 in Puglia, 661 cases of pertussis in subjects less than 18 years of age were identified in at least 1 data set (Fig, Supplemental Digital Content 1, http://links.lww.com/INF/C851 which shows the distribution of the cases in 2006–2013 and 2014–2015 according to the data source). Of the 661 cases, 47.7% were male and 52.3% were female. The age ranged from 0 to 17.4 years: the median age was 2.75 years (interquartile range: 0.25–8.9 years). Of the 661 patients, 40% were children younger than 1 year and 35% were younger than 6 months. The incidence of pertussis between 2006 and 2015 peaked in 2014 (2.56/100,000 inhabitants). The pertussis incidence in the following year (2015, the last year of the study period) was similar (2.54/100,000 inhabitants; Fig. 1). The incidences in 2014 and 2015 represented a doubling of the average incidence that had been recorded in the previous 8 years.
In terms of vaccine coverage for pertussis at 24 months of age in Puglia in 2006–2015, vaccination rates tended to decline over time (Fig. 1). The average vaccination rate during the study period was 96.4%, but from 2014 onwards, the rate dropped below the 95% threshold. The lowest vaccination rate was registered in 2015 (93.81%).
In 2006–2015, 80.3% (n = 531) of the 661 cases had to be hospitalized. Of these 531 cases, 45.4% were children who were less than 1 year of age (Fig, Supplemental Digital Content 2, http://links.lww.com/INF/C852 which shows the age distribution of the 661 hospitalized and not-hospitalized cases and the databases that provided these cases).
Figure 2 shows the incidence of pertussis in 2006–2015 in children who were less than 1 year of age. The average incidence was 74.9/100,000. The highest incidence was in 2015 (149.9/100,000). Figure 2 also shows the hospitalization rate of children <1 year of age in Puglia in 2006–2015. In 2006, the hospitalization rate was 0.59/1000. The highest hospitalization rate was in 2015 (1.16/1000). The mean length of hospitalization was 7 and 5 days for patients who were <1 and >1 year of age, respectively. This difference between age groups was statistically significant (P < 0.01).
RRL Pertussis Samples Subjected to Sequencing and MLVA
Between June 2014 and August 2016, 83 pertussis cases were confirmed by the RRL of the Puglia region. Of these cases, 57% were male and 43% were female (Table 1). The median age of these 83 cases was 4 (interquartile range: 2–36) months; the majority (59%, n = 49) were infants <6 months of age. The remaining 34 cases included 8 (9.7%) infants 7–12 months of age, 12 (14.5%) 1–4 year olds, 7 (8.4%) 5–9 year olds, 4 (4.8%) 10–14 year olds, 1 (1.2%) 15–18 year old and 2 (2.4%) >18 year olds. Of the 83 cases, 50 (60%) were hospitalized. Of these 50 hospitalized cases, 76% were infants <6 months of age.
Of the 83 pertussis cases, 21.7% were fully vaccinated, 21.7% were undervaccinated because the child was not old enough to receive all 3 doses, 8.4% were undervaccinated, 12% were unvaccinated and 33.7% were unvaccinated because the child was too young to receive the first dose (Table 1). The vaccination status was unknown for 2 cases (2.5%). Hospitalization associated significantly with age (P = 0.002) and the vaccination status (P = 0.007). Logistic regression analysis showed that infants <6 months of age were more likely to be hospitalized than older children (odds ratio=6.33; 95% confidence interval: 2.39–16.74). Moreover, infants who were unvaccinated because the child was too young to receive the first dose were more likely to be hospitalized than the children who were partially or fully vaccinated (odds ratio=5.1; 95% confidence interval: 1.47–17.62).
Molecular Characterization of BP
Of the 83 BP-positive samples that were collected in June 2014–August 2016, 70 underwent molecular characterization. The remaining 13 samples were not available. In 64 of these 70 samples (91.4%), the allelic profile was ptxA1-ptxP3-prn2. The remaining 6 samples had the ptxA1-ptxP3 profile but lacked the prn gene (4 because of the insertion of IS481 and 2 because of the introduction of a stop codon). These 6 samples were all from unvaccinated infants younger than 6 months of age: all were hospitalized with paroxysmal cough, postussive vomiting and apnea episodes. No epidemiologic linkage emerged among these cases.
Further analysis was performed on all 10 BP strains that were obtained during an outbreak in 2010. All were the same hypervirulent variant that predominated in the June 2014–August 2016 period.
MLVA was performed on 10 of the 83 BP-positive samples. These samples were randomly selected from the 62 samples that were obtained in 2015–2016. The preliminary MLVA analysis revealed a single MLVA type (MT), namely, MT27, in association with the ptxA1-ptxP3-prn2 profile.
Pertussis continues to be a relevant public health concern, in particular in newborn babies and unvaccinated and undervaccinated subjects. In Italy, pertussis vaccination coverage was very low until acellular vaccines were introduced in 1996. After the combined Diphtheria, Tetanus and Pertussis vaccine (DTaP) were introduced, vaccine coverage levels of over 95% were quickly reached. Starting in 2001, hexavalent vaccines have been the first choice product for infant immunization against pertussis, and their wide use has helped to maintain very high coverage levels. Despite these high coverage levels, however, Europe has recently experienced an apparent resurgence of pertussis. Possible contributing factors may be waning immunity, pathogen adaptation and increased awareness, and improved diagnostics.16
The Puglia region has a long tradition in the field of pertussis vaccination as it was one of the sites in the large clinical trial that was performed in Italy in 1991 to assess the effectiveness of the (at the time) new acellular vaccines.30 The vaccine coverage against pertussis in Puglia has always been consistent with the national average coverage.31 The present study of data from different sources showed that, in Puglia, the incidence of pertussis started rising in 2013. This trend has also been observed in other European countries.18 This study also showed that, during the 2006–2015 study period, the highest incidence of pertussis was in 2014. This high incidence was also observed in 2015. This increase in pertussis incidence occurred at the same time that the coverage at 24 months of age with the hexavalent combined vaccine fell below the threshold of 95%, which is considered the standard for achieving herd immunity.32 However, it is unlikely that the drop in vaccine coverage caused an increase in pertussis cases. Notably, the incidence of pertussis rose particularly sharply for children who were <1 year of age: in 2012, the incidence was 42/100,000 and it increased to 149/100,000 in 2015.
We also found that, in 2006–2015, 80% of the cases required hospitalization, and almost half (45%) of these children were less than 1 year of age. This finding is consistent with an Italian study that showed that 57% of the patients who were hospitalized for pertussis in 1999–2009 were less than 1 year old.15 It is also consistent with a report from Tuscany on pertussis hospitalization rates: in 2000–2013, 75% of the hospitalized cases were infants.33
These findings are confirmed by our separate analysis of the 83 BP-positive samples that were collected in June 2014–August 2016 by the RRL that serves the Puglia region. Since April 2014, the RRL in Bari collects the pharyngeal swabs from all hospitalized and nonhospitalized patients when there was a clinical suspicion of pertussis. Of the 83 cases, 60% required hospitalization and 76% of those were infants under the age of 6 months. Multivariate analysis also showed that these very young infants had a 6.33-fold greater risk of being hospitalized than the general infected population. Thus, these results confirm that the disease remains endemic in infants. It is also notable that a number of the cases in this study were fully vaccinated children.
Of the 83 BP-positive samples that were analyzed by the RRL in June 2014–August 2016, 70 were subjected to molecular characterization. The patients from whom these samples were obtained belonged to all age brackets. This is important because previous studies in Italy that characterized BP-positive samples focused exclusively on samples from infants.9,34,35 We found that all but 6 of the 70 samples had the variant ptxA1-ptxP3-prn2 allelic profile. The remaining 6 samples were prn-deficient BP strains. The ptxA1-ptxP3-prn2 variant was also detected in fully vaccinated children. This suggests that the increased ptx production of the mutant strain may increase disease severity in children who lack or have insufficient immunity. This was also suggested by other studies.16,36
The hypervirulent ptxA1-ptxP3-prn2 variant now circulates in many other European countries apart from Italy.16,35 Notably, European countries that use the acellular vaccine showed the circulation of the ptxA1-ptxP3-prn2 variant in 1998–2012.37 By contrast, Poland, which uses a whole cell vaccine, shows a different predominant BP profile, namely, ptxA1-ptxP1-prn1. This suggests that the acellular and whole cell vaccines may select different BP populations.37 This is supported by a study in China, which also uses the whole cell vaccine: the predominant BP allelic variant in China differs from the predominant strain that circulates in countries that use acellular vaccines.38
Six of our RRL samples from Puglia were prn-deficient BP strains. This allelic profile has already been observed to circulate in Italy.35Prn-deficient strains were first identified in 2000 in Finland, France, Japan and the United States of America.23 These strains were rarely found when whole cell vaccines were administered, namely, before the introduction of acellular vaccines.39 In several European countries, the frequency of prn-deficient strains is low (3.4%): exceptions are France and Norway.23 In Japan and the United States of America, where the acellular vaccines were introduced in 1981 and 1991, respectively, the proportion of circulating prn-deficient BP strains is higher than in Europe.23
Recently, MT27 was found to be the predominant MLVA profile in Australia,40 Europe,29 the United States of America41 and Japan.42 MT27 associates with the prn2 and ptxP3 profile that was identified in the samples from Puglia. This MT has been found all over the world and has the potential to cause epidemics as a result of positive selection in a highly vaccinated population.29 The fact that we and others35 only identified a few BP molecular profiles in Italy suggests that there may have been clonal diffusion of BP throughout the country. However, given the low number of Italian samples that have been analyzed by MLVA, it is also possible that this low variation is due to selection bias.
Although the incidence of pertussis in Italy decreased after the introduction of immunization,3 the present study found that the disease has resurged in recent years, especially in children less than 1 year of age. It seems unlikely that this resurgence is because of the slight decline in vaccination coverage that was observed in 2014–2015. It is much more likely that the cause of the resurgence is the lack of a comprehensive booster vaccination strategy, including vaccination of pregnant women. Another likely cause of the resurgence is the loss of vaccine efficacy because the current vaccine has placed selection pressure on the circulating BP strains.43 The presence of the hypervirulent BP variant in children who have been fully vaccinated (ie, they have undergone their primary vaccination course and have received booster doses) suggests that bacterial adaptation has likely played a role in the resurgence of pertussis.2 This notion is supported by the fact that pertussis has re-emerged in Europe after the acellular vaccine replaced the whole cell vaccine.44
To limit BP infections and protect unvaccinated infants, it has been recommended that the mother and all family members who may have close contact with the newborn should be vaccinated: this is called the cocoon strategy.5 However, this strategy still does not protect infants from possible transmission from colonized vaccinated asymptomatic family members and others.45,46 Moreover, several studies suggest that administering the pertussis vaccine during pregnancy may weaken the response of the infant to the primary immunization cycle.47 To prevent this, it is currently recommended that the mother should be immunized during the third trimester of pregnancy; this recommendation was also reiterated in the new approved National Plan of Vaccine Immunization.32,48,49 Moreover, this strategy seems to be the only cost-effective strategy of preventing pertussis infections in infants.50,51
The findings of the present study suggest that enhanced surveillance of pertussis and systematic laboratory confirmation of cases are needed in Italy. Only then will we be able to assess the real epidemiology of the disease and the emergence of new strains that can escape the immunity that is induced by vaccination. Monitoring the circulating strains is also crucial for designing future vaccination strategies and informing research on new pertussis vaccines.
We thank Maria Giovanna Cappelli and Davide Parisi for data collection and Donatella Pepe and Daniele Casulli for technical assistance in the laboratory investigations.
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