Laboratory Diagnosis of Pertussis.

The introduction of vaccination in the 1950s significantly reduced the morbidity and mortality of pertussis. However, since the 1990s, a resurgence of pertussis has been observed in vaccinated populations, and a number of causes have been proposed for this phenomenon, including improved diagnostics, increased awareness, waning immunity, and pathogen adaptation. The resurgence of pertussis highlights the importance of standardized, sensitive, and specific laboratory diagnoses, the lack of which is responsible for the large differences in pertussis notifications between countries. Accurate laboratory diagnosis is also important for distinguishing between the several etiologic agents of pertussis-like diseases, which involve both viruses and bacteria. If pertussis is diagnosed in a timely manner, antibiotic treatment of the patient can mitigate the symptoms and prevent transmission. During an outbreak, timely diagnosis of pertussis allows prophylactic treatment of infants too young to be (fully) vaccinated, for whom pertussis is a severe, sometimes fatal disease. Finally, reliable diagnosis of pertussis is required to reveal trends in the (age-specific) disease incidence, which may point to changes in vaccine efficacy, waning immunity, and the emergence of vaccine-adapted strains. Here we review current approaches to the diagnosis of pertussis and discuss their limitations and strengths. In particular, we emphasize that the optimal diagnostic procedure depends on the stage of the disease, the age of the patient, and the vaccination status of the patient.

Estimation of household transmission rates of pertussis and the effect of cocooning vaccination strategies on infant pertussis.

BACKGROUND: Despite >50 years of universal vaccination, pertussis remains the most prevalent vaccine-preventable infectious disease in developed countries. Pertussis is often mild in adults, but can run a severe course in young infants. METHODS: Data on transmission of pertussis within households were captured in a population-based, nationwide, prospective study performed in the Netherlands between February 2006 and December 2009. We estimated the transmission rates of pertussis with a clinically confirmed infection in 140 households, using stochastic epidemic models. Parameter estimates were used to gauge the effect of vaccinating household members (cocooning) to prevent the infection in young infants. RESULTS: Overall transmission rates in the household were high. Fathers were less susceptible than other household members (estimated relative susceptibility of fathers = 0.44 [95% confidence interval (CI) = 0.27-0.72]), whereas mothers may be more infectious to their infants than are other household members (estimated relative infectiousness of mothers = 3.9 [95% CI = 0.59-14]). Targeted vaccination of mothers would approximately halve the probability of infants' infection. Vaccination of siblings is less effective in preventing transmission within the household, but may be as effective overall because siblings more often introduce an infection in the household. Vaccination of fathers is expected to be least effective. CONCLUSIONS: Selective vaccination of persons in households with a young infant may substantially reduce the disease burden of pertussis in infants by reducing transmission within the household.

Two-component cluster analysis of a large serodiagnostic database for specificity of increases of IgG antibodies against pertussis toxin in paired serum samples and of absolute values in single serum samples.

Measuring IgG antibodies against pertussis toxin (IgG-Ptx) with an enzyme-linked immunosorbent assay (ELISA) can be used to diagnose pertussis infection; however, the cutoff points are not unanimously defined. To determine the diagnostic specificity of increases of IgG-Ptx in paired sera and of absolute values in single serum samples, we applied a two-component cluster analysis to serum samples of patients suspected for pertussis, whose sera had been submitted to a routine diagnostic laboratory between 2003 and 2009, and had been assayed with an in-house IgG-Ptx ELISA calibrated with the international FDA lot 3 IgG-Ptx reference serum. Children eligible for the acellular pertussis vaccination were excluded to avoid interference from a vaccine-induced IgG-Ptx rise. Binary distribution mixtures were fitted to the data. Receiver operating characteristic (ROC) curves were calculated for absolute values in single samples (n = 14,452) and increases in paired samples (n = 2,455). For both parameters, two subpopulations could be identified: a population with high reactivity (persons with pertussis infection) and a population with low reactivity (persons without pertussis infection). For absolute values in single samples, the area under the curve (AUC) of the ROC curve was 0.993 and the optimum cutoff (with the highest cumulative value of specificity plus sensitivity) was 67.7 IU/ml (95% confidence interval, 63.9 to 74.1; sensitivity, 96.4%; specificity, 95.7%). A previously determined diagnostic cutoff of 125 IU/ml was associated with a sensitivity of 88.1% and a specificity of 98.8%. For increases in paired sera, the AUC was 0.999 and the optimum cutoff was 3.1-fold (95% CI, 2.8 to 3.4; sensitivity, 99.6%; specificity, 99.2%). Given the methodology of this study, estimates of sensitivity probably are overrated (because pertussis patients without IgG-Ptx response are not detected), but estimates of specificities can be considered very accurate.

Seroprevalence of pertussis in The Netherlands: evidence for increased circulation of Bordetella pertussis.

BACKGROUND: In many countries, the reported pertussis has increased despite high vaccination coverage. However, accurate determination of the burden of disease is hampered by reporting artifacts. The infection frequency is more reliably estimated on the basis of the prevalence of high IgG concentrations against pertussis toxin (IgG-Ptx). We determined whether the increase in reported pertussis in the last decade is associated with an increase in the number of infections. METHODOLOGY/PRINCIPAL FINDINGS: In a cross-sectional population-based serosurveillance study conducted in 2006-07, from a randomly selected age-stratified sample of 7,903 persons, serum IgG-Ptx concentrations were analyzed using a fluorescent bead-based multiplex immuno assay. In 2006-07, 9.3% (95%CI 8.5-10.1) of the population above 9 years of age had an IgG-Ptx concentration above 62.5 EU/ml (suggestive for pertussis infection in the past year), which was more than double compared to 1995-96 (4.0%; 95%CI 3.3-4.7). The reported incidence showed a similar increase as the seroprevalence between both periods. CONCLUSIONS: Although changes in the vaccination program have reduced pertussis morbidity in childhood, they have not affected the increased infection rate in adolescent and adult pertussis. Indeed, the high circulation of B. pertussis in the latter age-categories may limit the effectiveness of pediatric vaccination.

Cost-effectiveness of adolescent pertussis vaccination for the Netherlands: using an individual-based dynamic model.

BACKGROUND: Despite widespread immunization programs, a clear increase in pertussis incidence is apparent in many developed countries during the last decades. Consequently, additional immunization strategies are considered to reduce the burden of disease. The aim of this study is to design an individual-based stochastic dynamic framework to model pertussis transmission in the population in order to predict the epidemiologic and economic consequences of the implementation of universal booster vaccination programs. Using this framework, we estimate the cost-effectiveness of universal adolescent pertussis booster vaccination at the age of 12 years in the Netherlands. METHODS/PRINCIPAL FINDINGS: We designed a discrete event simulation (DES) model to predict the epidemiological and economic consequences of implementing universal adolescent booster vaccination. We used national age-specific notification data over the period 1996-2000--corrected for underreporting--to calibrate the model assuming a steady state situation. Subsequently, booster vaccination was introduced. Input parameters of the model were derived from literature, national data sources (e.g. costing data, incidence and hospitalization data) and expert opinions. As there is no consensus on the duration of immunity acquired by natural infection, we considered two scenarios for this duration of protection (i.e. 8 and 15 years). In both scenarios, total pertussis incidence decreased as a result of adolescent vaccination. From a societal perspective, the cost-effectiveness was estimated at €4418/QALY (range: 3205-6364 € per QALY) and €6371/QALY (range: 4139-9549 € per QALY) for the 8- and 15-year protection scenarios, respectively. Sensitivity analyses revealed that the outcomes are most sensitive to the quality of life weights used for pertussis disease. CONCLUSIONS/SIGNIFICANCE: To our knowledge we designed the first individual-based dynamic framework to model pertussis transmission in the population. This study indicates that adolescent pertussis vaccination is likely to be a cost-effective intervention for The Netherlands. The model is suited to investigate further pertussis booster vaccination strategies.

Impact of acellular pertussis preschool booster vaccination on disease burden of pertussis in The Netherlands.

BACKGROUND: An acellular preschool booster vaccination against pertussis has been included in the National Immunization Programme in The Netherlands, since November 2001. We studied the impact of this preschool booster on the epidemiology of pertussis. METHODS: We analyzed and compared pertussis cases registered in the national notification system, hospital registry, and death registry between the periods 1998-2001 (without preschool booster) and 2002-2005 (with preschool booster). RESULTS: In 2002-2005, the incidence of hospitalizations and notifications in 1-4 year olds were 48% and 44%, lower respectively, than in 1998-2001. Similarly, in 5-9 year olds the incidence of hospitalizations and notifications had decreased 32% and 15%, respectively. In 2005, vaccine effectiveness for preschool booster among children born between January 1, 1998 and January 1, 2001--all of whom had been eligible for the booster--was estimated at 79% (95% CI: 71-85). In infants aged 0-6 months, the incidence of hospitalizations per 100,000 population decreased 40%, from 222.5 to 133.6. In contrast, among cohorts aged 10-19, 20-59, and >60 years, the incidence of notifications increased 60%, 44%, and 68%, respectively. CONCLUSIONS: The preschool booster strongly decreased the disease burden in the targeted cohorts. Importantly, the incidence in infants 0-6 months also showed a decline after introduction of the preschool booster, suggesting reduced transmission from siblings to young infants. Meanwhile, the number of pertussis cases in adolescents and adults increased. With prevention of severe pertussis among infants as focus, this effect should not be ignored in the discussion on future vaccination strategies for pertussis.

Sensitivity of three serum antibody tests in a large outbreak of Legionnaires' disease in the Netherlands.

In 1999, an outbreak involving 188 patients with Legionnaires' disease (LD) occurred at a flower show in the Netherlands. This large outbreak provided the opportunity to evaluate serum antibody tests to assay anti-Legionella pneumophila, since limited data are available on the sensitivity of these tests. The sensitivities of an indirect serotype 1-6 immunofluorescence antibody test (IFAT), a rapid micro-agglutination test (RMAT) IgM serotype 1 antibody assay, and an ELISA to detect IgM and IgG serotype 1-7 antibodies, were evaluated using serum samples from LD patients related to the 1999 outbreak. Sensitivity was calculated using positive culture and/or a positive urinary antigen test as the gold standard in outbreak-related patients with radiographically confirmed pneumonia who fulfilled the epidemiological criteria. The IFAT, RMAT and ELISA showed sensitivities of 61, 44 and 64%, respectively. The sensitivity of the three tests combined was 67%. In epidemic situations, however, high standing titres may be included in the laboratory evidence of LD cases. In the study population, high standing titres were found in 16% of cases. If the presence of high standing antibody titres was added to the criteria of a positive test, the sensitivities of IFAT, RMAT and ELISA were 86, 48 and 75%, respectively. The sensitivity was 91% for all tests combined. The higher sensitivity for the combined use of tests is offset by a reduction in specificity to 97.6%. The results of this study indicate that using a combination of serologic tests in pneumonia patients suspected to have LD does not substantially improve sensitivity. The results suggest that in the microbiological diagnosis of LD, both IFAT and ELISA are reasonably sensitive assays. In an epidemic situation, both tests are highly sensitive, the IFAT more so than the ELISA.

The incidence of Bordetella pertussis infections estimated in the population from a combination of serological surveys.

OBJECTIVES: Bordetella pertussis circulates even in highly vaccinated populations. There is a considerable amount of infection in adults. For designing more effective vaccination schedules it is important to quantify the age-dependent relation between the number of notified cases and the number of infections. METHODS: We used a statistical relationship between the time since infection and the IgG antibody titers against pertussis toxin, derived from a longitudinal data set, to estimate time since infection for all individuals in a cross-sectional population-based study (1995-1996) based on their titers. Age-specific incidence of infection with B. pertussis was calculated and compared with the age-distribution of notified cases of pertussis in 1994-1996. RESULTS: Estimated incidence of infection was 6.6% per year for 3-79-year olds, annual incidence of notified cases 0.01%. Estimated age-specific incidence of infection was lowest for 3-4-year olds (3.3%) and increased gradually up to the age of 20-24 years (10.8%). The number of notified cases was highest for 3-9-year olds. CONCLUSIONS: In the Dutch population B. pertussis infections occur more frequently and in elder age-categories then suggested by notifications. Mathematical modeling could explore what booster vaccination strategies are most effective in reducing severe disease among not (completely) vaccinated infants.

Pathogen adaptation under imperfect vaccination: implications for pertussis.

Mass vaccination campaigns have drastically reduced the burden of infectious diseases. Unfortunately, in recent years several infectious diseases have re-emerged. Pertussis poses a well-known example. Inspired by pertussis, we study, by means of an epidemic model, the population and evolutionary dynamics of a pathogen population under the pressure of vaccination. A distinction is made between infection in immunologically naive individuals (primary infection) and infection in individuals whose immune system has been primed by vaccination or infection (secondary infection). The results show that (i) vaccination with an imperfect vaccine may not succeed in reducing the infection pressure if the transmissibility of secondary infections is higher than that of primary infections; (ii) pathogen strains that are able to evade the immunity induced by vaccination can only spread if escape mutants incur no or only a modest fitness cost and (iii) the direction of evolution depends crucially on the distribution of the different types of susceptibles in the population. We discuss the implications of these results for the design and use of vaccines that provide temporary immunity.

Pertussis sources of infection and routes of transmission in the vaccination era.

Vaccination against pertussis has resulted in reduction of the infection pressure of Bordetella pertussis (partial herd immunity), but the circulation of B. pertussis has persisted as a consequence of waning of vaccine-induced and naturally acquired immunity. An increase in the reported incidence of B. pertussis infection in older children, adolescents and young adults has been noted, resulting in a perceived resurgence of the disease in these age groups. Regardless of whether this resurgence is real or not, older groups are increasingly recognized as playing an important role in transmitting B. pertussis infection to incompletely immunized infants, in whom pertussis disease continues to cause severe and fatal illness, albeit at much lower levels than in the prevaccine era. Several studies have suggested that mothers, in particular, are a significant source of infection for infants. Adolescents, grandparents and health care workers can also play a role. By contrast, most adolescents acquire the infection from schoolmates and friends, whereas for adults the main sources are children and work colleagues. Furthermore teachers, child care workers and health care workers could be at increased risk of being exposed to, and transmitting, B. pertussis infection. Current immunization strategies inadequately control the circulation of B. pertussis, in part because of suboptimal adherence to current pediatric immunization guidelines. In addition to efforts to improve pertussis immunization rates in children, the expansion of pertussis immunization to target specific groups should be considered. Besides reducing morbidity in the targeted groups, these strategies could decrease the residual burden of pertussis morbidity and mortality in infants.

New pertussis vaccination strategies beyond infancy: recommendations by the global pertussis initiative.

BACKGROUND: The Global Pertussis Initiative, an expert scientific forum, was established to address the ongoing problems associated with pertussis disease worldwide. METHODS: The group analyzed pertussis disease trends, developed recommendations to improve disease control through expanded vaccination strategies, and proposed solutions to barriers to implementation and support of research activities. RESULTS: Bordetella pertussis infection is endemic and continues to be a serious problem among unvaccinated or incompletely vaccinated infants. In addition, the reported incidence of pertussis disease is increasing in adolescents and adults, who not only experience a considerable health burden themselves but also infect vulnerable infants. CONCLUSIONS: Current vaccination strategies need to be reinforced. Expanded vaccination should include adding booster doses to existing childhood schedules (preschool or adolescent) and booster doses for those specific adult subgroups that have the highest risk of transmitting B. pertussis infection to infants (i.e., new parents, other contacts of newborns, and health care workers). More epidemiological studies and studies of disease transmission and the cost-effectiveness of vaccination would be valuable, and surveillance, diagnostic improvements, and educational campaigns are needed for implementation. However, as a prelude to universal adult vaccination, immediate universal adolescent vaccination should be instituted in countries in which it is economically feasible.

Estimating the incidence of subclinical infections with Legionella Pneumonia using data augmentation: analysis of an outbreak in The Netherlands.

Infections with Legionella bacteria can cause a potentially lethal form of pneumonia known as legionnaires' disease. In 1999 a major outbreak, causing 31 deaths, occurred among visitors and exhibitors of a consumer fair in The Netherlands. The epidemiology of subclinical infections is largely unknown, as there is no reliable method to diagnose such infections. To explore the incidence of subclinical infections, IgG and IgM antibody levels among exhibitors were compared to those among a representative sample of the Dutch population. As exhibitors were assumed to comprise both infected and uninfected individuals, their antibody levels were modelled as a mixture distribution. As infected individuals are expected to cluster around a point source, the spatial aspect of the spread of infections was taken into account. To estimate the distribution of antibody levels among infected individuals and to impute infection status among exhibitors, data augmentation was used. Subclinical infection appeared to be very common and its frequency declined with the distance from the putative source of the outbreak.

Site-specific manifestations of invasive group a streptococcal disease: type distribution and corresponding patterns of virulence determinants.

As part of a national surveillance program on invasive group A streptococci (GAS), isolates that caused specific manifestations of invasive GAS disease in The Netherlands were collected between 1992 and 1996. These site-specific GAS infections involved meningitis, arthritis, necrotizing fasciitis, and puerperal sepsis. An evaluation was performed to determine whether GAS virulence factors correlate with these different disease manifestations. PCRs were developed to detect 9 genes encoding exotoxins and 12 genes encoding fibronectin binding proteins. The genetic backgrounds of all isolates were determined by M genotyping and pulsed-field gel electrophoresis (PFGE) analysis. The predominant M types included M1, M2, M3, M4, M6, M9, M12, and M28. Most M types were associated with all manifestations of GAS disease. However, M2 was found exclusively in patients with puerperal sepsis, M6 predominated in patients with meningitis, and M12 predominated in patients with GAS arthritis. While characteristic gene profiles were detected in most M types, the resolution of detection of different gene profiles within M genotypes was enhanced by PFGE analysis, which clearly demonstrated the existence of some clonal lineages among invasive GAS isolates in The Netherlands. M1 isolates comprised a single clone carrying highly mitogenic toxin genes (speA, smeZ) and were associated with toxic shock-like syndrome. Toxin profiles were highly conserved among the most virulent strains, such as M1 and M3.

Legionnaires' disease at a Dutch flower show: prognostic factors and impact of therapy.

After a large outbreak of Legionnaires' disease in the Netherlands, we determined risk factors for intensive care unit (ICU) admission and death and the impact of adequate therapy on ICU-free survival among 141 hospitalized patients. Overall mortality rate was 13%, and ICU mortality rate was 36%. Smoking, temperature >38.5 degrees C, and bilateral infiltrates shown on chest x-ray were independent risk factors for ICU admission or death (all p<0.05). Starting adequate therapy within 24 hours after admission resulted in a higher ICU-free survival rate compared to therapy initiation after 24 hours: 78% versus 54%, respectively (p=0.005). However, delay in providing therapy to patients with urinary antigen tests with negative results did not influence outcome. These data suggest that by using the urinary antigen test on admission a more tailored approach to patients with community-acquired pneumonia may be applied.

Sensitivity of three urinary antigen tests associated with clinical severity in a large outbreak of Legionnaires' disease in The Netherlands.

In 1999 an outbreak involving 188 patients with Legionnaires' disease (LD) occurred among visitors to a flower show in the Netherlands. Two enzyme immunoassays (Binax and Biotest) and one immunochromatographic assay (Binax NOW) were tested, using urine samples from LD patients from the 1999 outbreak. Sensitivity was calculated using positive culture and/or seroconversion as the "gold standard" in outbreak-related patients with radiographically confirmed pneumonia who fulfilled the epidemiological critera. The Binax EIA, Biotest EIA, and Binax NOW assay showed overall sensitivities of 69, 71, and 72%, respectively. When the tests were performed with concentrated urine samples, the overall sensitivities increased to 79, 74, and 81%, respectively. Using multiple logistic regression analysis with backward elimination, a statistically significant association was found between clinical severity and test sensitivity for all tests. For patients with mild LD, the test sensitivities ranged from 40 to 53%, whereas for patients with severe LD who needed immediate special medical care, the sensitivities reached 88 to 100%. These findings have major implications for the diagnostic process in patients with mild pneumonia and suggest that patients with mild pneumonia may go underdiagnosed if urine antigen tests alone are used.

A large outbreak of Legionnaires' disease at a flower show, the Netherlands, 1999.

In 1999, an outbreak of Legionnaires' disease affected many visitors to a flower show in the Netherlands. To identify the source of the outbreak, we performed an environmental investigation, as well as a case-control study among visitors and a serologic cohort study among exhibitors to measure exposure to possible sources. Of 77,061 visitors, 188 became ill (133 confirmed and 55 probable cases), for an attack rate of 0.23% for visitors and 0.61% for exhibitors. Two whirlpool spas in halls 3 and 4 of the exhibition and a sprinkler in hall 8 were culture positive for Legionella pneumophila. One of three genotypes found in both whirlpool spas was identical to the isolates from 28 of 29 culture-positive patients. Persons who paused at the whirlpool spa in hall 3 were at increased risk for becoming ill. This study illustrates that whirlpool spas may be an important health hazard if disinfection fails.

Detection of Borrelia afzelii, Borrelia burgdorferi sensu stricto, Borrelia garinii and group VS116 by PCR in skin biopsies of patients with erythema migrans and acrodermatitis chronica atrophicans.

OBJECTIVE: To evaluate the diagnostic performance of two polymerase chain reaction (PCR) procedures using skin biopsies of 20 erythema migrans (EM) and 24 acrodermatitis chronica atrophicans (ACA) patients. METHODS: One assay amplified a fragment of the outer surface protein (Osp) A gene. The second method amplified the spacer region between the 5S and 23S rRNA genes; hybridization of this fragment allowed identification of Borrelia burgdorferi sensu lato species. RESULTS: Among EM patients, both assays detected Borrelia DNA in 15 samples. Among ACA patients, the ospA PCR detected 15 positives and 10 samples were positive by 5S-23S PCR. In 19 samples one species was detected, 15 skin biopsies contained Borrelia afzelii, and Borrelia garinii was found in two patients. Group VS116 was detected in two EM patients, and therefore this group has pathogenic potential. Mixed infections of B. afzelii and B. garinii, group VS116 or B. burgdorferi sensu stricto were found in three EM and three ACA patients. CONCLUSIONS: Diagnosis of EM and ACA by PCR is useful and knowledge of the presence of species may be used to predict the course of disease or the need for further antibiotics.