Bordetella pertussis produces a number of virulence determinants believed to contribute to its survival in the host as well as to the pathogenesis of disease. One of these factors, adenylate cyclase toxin (ACT), has been implicated to penetrate human neutrophils and macrophages and abrogate their function by virtue of unregulated production of intracellular cAMP. In order to adequately study the nature of ACT and its role in pathogenesis, it is necessary to isolate the toxin from other virulence factors produced by the organism. Attempts by other investigators to purify ACT and maintain both its invasive and catalytic properties have not been successful. B. pertussis produces a cell associated ACT during mid-log phase of growth in Stainer-Scholte medium. Purification of ACT with both activities from urea extracted whole cells has been achieved by hydroxylapatite and calmodulin-sepharose chromatography. ACT is a single protein of 220 kd molecular weight with an isoelectric point of 7.0. The protein probably contains regions which are strongly hydrophobic. ACT has a specific activity of nearly 17,000 μM cAMP formed/min. An 850 ng sample of ACT induced over 1,400 pmoles cAMP/10⁶ S49 mouse lymphoma cells while 660 ng of ACT inhibited human neutrophil chemiluminescence by 65%.

Objective: Describe the prevalence of Bordetella pertussis via PCR in children under 5 years old hospitalized as probable cases of pertussis and report the most common clinical features among them. Results: A positive PCR result for B. pertussis was observed in 20.5% of our samples (18/88), one-third of them were from infants between 2 and 3 months old. The most common symptoms were paroxysms of coughing (88.9%), difficulty breathing (72.2%), cyanosis (77.8%) and fever (50%). The mother was the most common symptomatic carrier (27.8%), followed by uncles/aunts (22.2%) among children with pertussis. / This work was supported by fourth research incentive of the Universidad Peruana de Ciencias Aplicadas (UPC), Lima‑Peru. / Revisión por pares

ASTMH 64th Annual Meeting. October 25-29, 2015 Philadelphia Marriott Downtown Philadelphia, Pennsylvania USA / Acute respiratory infections (ARI) are the main cause of morbidity and mortality in children under 5 years worldwide. Bordetella pertussis is a highly contagious bacterium that can cause serious illness, and approximately half of infected infants less than 1 year old are hospitalized. Also, pertussis immunization series is not completed until six months of age, leaving young infants vulnerable to pertussis. In Peru, pertussis is an increasing health problem despite immunization efforts, and the role of B. pertussis in ARI is unknown. We determined the prevalence of B. pertussis among children under 5 years old admitted to Hospital Nacional Cayetano Heredia in Lima with diagnosis of ARI between Jan-2009 and Dec 2010. Epidemiological and clinical features were collected, and presence of B. pertussis was determined by PCR (pertussis toxin and IS481 gene). A total of 596 nasopharyngeal samples among children under 5 years were analyzed. In 114 (19.1%) samples were positive for B. pertussis. 32.5% of sample positive to B. pertussis were diagnosed as viral pneumonia at diagnosis. Importantly, 71.9% of cases were under 12 months of age and 58.8% have been contact with other ARI infected people. Significant differences in clinical symptoms between the total ARI cases and B. pertussis cases were not found. The most frequent symptoms in B. pertussis cases were fever (100%), rhinorrhea 78%, cough 71.9% and respiratory distress 60.5%. One child died due to the infection. B. pertussis cases showed a seasonal distribution with peaks during the months March June and November. This study shows the high prevalence of B. pertussis in infants who were hospitalized due to severe acute respiratory infections in Lima, Peru. Epidemiologic surveillance programs for B. pertussis are essential in the future in Peru

joruiz@clinic.ub.es / Objective: To characterize two Achromobacter xylosoxidans recovered from 2 patients diagnosed with pertussis during a Bordetella pertussis surveillance program. Methods: Nasopharyngeal swabs from 2 children under 1 year of age with clinical suspicion of pertussis were analyzed by culture and PCR. Results: Two Achromobacter xylosoxidans A8, closely related to Bordetella spp. were recovered from 2 patients diagnosed of pertussis, both carrying the ptxA gene and IS418 the pertussis toxin encoding gene. Subsequently, antibiotic susceptibility was evaluated by disk-diffusion method and by PCR. Conclusions: Although more detailed studies are needed, the present data highlight the possibility that Achromobacter xylosoxidans, closely related Bordetella pertussis microorganisms and not covered under the vaccine umbrella, might also result in cases of whooping cough. Thereby further surveillance is necessary to determine the extension and relevance of their pathogenic role in order to discriminate their real public health implication.

Dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Master of Science in Medicine. Johannesburg, 2015 / Pertussis, caused by Bordetella pertussis, is a vaccine-preventable disease affecting persons of all ages. Despite vaccination with either the whole-cell or acellular vaccine, the burden of pertussis has increased worldwide. The acellular vaccine was licensed in South Africa in 2009, replacing the whole-cell vaccine; however, due to no active surveillance, pertussis is underestimated in this country. This study describes the burden of disease caused by B. pertussis and other Bordetella species in patients with severe respiratory illness (SRI), influenza-like illness (ILI) and controls. Prospective, active surveillance was conducted amongst SRI and ILI patients and controls at two sentinel sites in South Africa. Patients who met the case definitions were enrolled from May 2012 to October 2014. Clinical and demographic data were collected. Induced sputum was collected from SRI patients only and combined nasopharyngeal/oropharyngeal specimens were collected from all patients and controls. Real-time polymerase chain reaction (PCR) was used to target the insertion sequences IS481, pIS1001, hIS1001 and pertussis toxin gene ptxS1. All data were analysed in Microsoft Excel (Microsoft Corporation). Statistical significance was determined using the chi-squared test and univariate logistic regression at p <0.05 for all parameters. Of 8569 cases that were enrolled and tested, 118 [1.4%, 118/8569 (95% CI 1.1 – 1.6)] were positive for B. pertussis of which 2% [80/3982 (95% CI 1.6 – 2.5)] presented with SRI, 1% [32/3243 (95% CI 0.7 – 1.4)] with ILI and 0.4% [6/1344 (95% CI 0.2 – 1.0)] were asymptomatic. Positive cases were stratified into confirmed pertussis and probable pertussis based on cycle threshold (Ct) value cut-offs generated by real-time PCR for IS481. Within the SRI population, there were more probable than confirmed pertussis cases [51/3982, 1.3% vs. 29/3982, 0.7%; p=0.02] and within the ILI group there were 0.5% confirmed and probable cases, respectively [15/3243, 0.5% vs. 17/3243, 0.5%; p=0.86]. The highest detection rate of pertussis in SRI positive cases was in the ≥65 year olds (2.8%, 6/208) and for the ILI positive cases the highest detection rate was in the 1-4 year olds (1.5%, 9/614). Pertussis disease was observed mainly in the winter and spring months with a 15% increase in disease detected in August 2014. The B. pertussis attributable fraction was 67% (95% confidence interval [CI] 18.49 – 86.63) for SRI positive cases. Fifty-eight percent (46/80) of B. pertussis positive cases were co-infected with respiratory bacteria (Streptococcus pneumoniae, Haemophilus influenzae, Legionella spp. or Mycoplasma pneumoniae) or viruses (influenza, respiratory syncytial virus (RSV), human metapneumovirus or other viruses (adenovirus, enterovirus, parainfluenza or rhinovirus). HIV status and full pertussis vaccination for age did not affect B. pertussis positivity. B. parapertussis was detected in 1% [40/3982 (95% CI 0.7 – 1.4)] of the SRI population, 0.6% [18/3243 (95% CI 0.3 – 0.9)] of the ILI population and in 0.1% [2/1344 (0.02 – 0.5)] of asymptomatic individuals. The highest detection rate for the SRI (1.6%, 8/497) and ILI (1.5%, 9/614) positive cases were in the 1-4 year olds. The B. parapertussis attributable fraction was 80% (95% confidence interval [CI] 12.52 – 95.38) for SRI cases. Four cases tested positive for B. bronchiseptica, of which one individual was HIV positive. B. pertussis, B. parapertussis and B. bronchiseptica were detected despite the case definitions not being ideal for the detection of these pathogens. Bordetella spp. was detected in all age groups tested. This study generates baseline data for pertussis in South Africa and surveillance is ongoing.

A coqueluche é uma doença respiratória contagiosa, causada pela bactéria Bordetella pertussis. Tendo um significativo impacto epidemiológico, esta doença sofreu uma expressiva redução após o uso disseminado de vacinas pertussis. Efeitos adversos na imunização com a Vacina de células inteiras Whole cell pertussis (wP), atribuídos a presença de lipopolissacarídeos (LPS), levou ao desenvolvimento da Vacina pertussis acelular (aP). Contudo, a imunização com aP não tem demonstrada a mesma eficiência que a imunização com wP. Diante desse cenário o objetivo deste trabalho é desenvolver o processo de produção industrial de uma nova Vacina Pertussis de células inteiras com reduzida quantidade de LPS e baixa reatogenicidade, a Vacina Pertussis Low (wPlow). Para alcançar o objetivo, foram produzidos em escala industrial 25 lotes de cultivos inativados, concentrados e submetidos à extração de LPS com solvente orgânico. Para extração de LPS foram avaliadas 4 diferentes metodologias: filtração de fluxo tangencial (TFF); filtração de fluxo tangencial com lavagem com solução com solvente orgânico (TFFSW); centrifuga tubular (CT); centrifugação de fluxo contínuo de pratos (CFC). A wPlow produzida em centrifuga de bancada e a wP foram usadas para comparação. O processo de bancada resultou na redução de LPS (método Purpald) em média de 75% do conteúdo de LPS e a redução de 81% da atividade endotóxica (dosagem de LAL). Nos processos industriais, por TFF houve a redução de &cong;21% do conteúdo de LPS, porém com aumento de &cong; 52% da atividade endotóxica; por TFFSW houve a redução de 46% do conteúdo de LPS e uma redução endotóxica média de &cong; 24%; por CT ocorreu à redução de &cong; 66% do conteúdo de LPS e de &cong; 73% da atividade endotóxica; com a CFC houve a redução de &cong; 92% do conteúdo de LPS e de &cong; 61% da atividade endotóxica. Os rendimentos de processo foram &cong; 83%, 61%, 37% e 63% respectivamente para os processos de TFF, TFFSW, CT e CFC. Através de microscopia eletrônica, foi possível visualizar a integridade celular após o processamento por CFC, e o principal antígeno vacinal, a toxina pertussis foi detectada na preparação, por western-blot. Quanto à imunogenicidade, anticorpos IgG anti-pertussis foram detectados por ELISA e resultados preliminares não mostraram diferença significativa de redução de colonização pulmonar de B. pertussis em camundongos imunizados com a wPlow ou wP. Diante dos resultados obtidos, podemos concluir que os processos de TFF e TFFSW não foram eficientes na remoção da atividade endotóxica da wPlow, embora tenha ocorrido a redução do LPS. Com relação aos processos utilizando centrífugas industriais, eficientes tanto na remoção do LPS e na redução da atividade endotóxica, houve, contudo, um baixo rendimento no processo com CT. A wPlow produzida por CFC foi imunogênica, indicando a eficácia potencial desta vacina e que a produção em escala industrial é um processo viável. Como parte deste trabalho, a cepa vacinal de Bordetella pertussis foi também caracterizada pelo seu sequenciamento genômico completo (genbank número CP010323). / Whooping cough is a contagious respiratory disease caused by Bordetella pertussis. In the past this infection had a high epidemiological impact, only reduced after the use of pertussis vaccine. The association of adverse events in immunization with whole cell Pertussis vaccine (wP), attributed to the presence of lipopolysaccharides (LPS), has led to the development of acellular Pertussis vaccine (aP). However, it is known that immunization with aP does not have the same efficiency as compared to immunization with wP vaccine. In this scenario, the objective of this work is to develop the industrial production process of a new whole cell pertussis vaccine with reduced amount of LPS and low reatogenicity, the whole cell Pertussis low vaccine (wPlow). To achieve this aim, we produced 25 lots of inactivated and concentrated cultures prepared on an industrial scale and subjected to LPS extraction with organic solvent. We evaluated four industrial processes to extract the LPS from the cells: tangential flow filtration (TFF), TFF with organic solvent washing (TFFSW), tubular centrifugation (CT) and continuous flow centrifugation (CFC). These methodologies were compared with wPlow produced at bench scale obtained by centrifugation and with traditional wP. The bench process resulted in the reduction of 75% of LPS content (Purpald method) and 81% reduction in endotoxic activity (LAL dosage) on average. In the industrial processes, TFF reduced &cong; 21% in LPS content, but with a &cong;52% increase in endotoxic activity; by TFFSW there was a reduction of &cong; 46% of the LPS content and an average reduction of endotoxic activity of &cong;24%; CT reduced &cong; 66% of LPS content and &cong; 73% of endotoxic activity; with CFC there was a reduction of &cong; 92% in LPS content and &cong;1% in endotoxic activity. The process yields were &cong; 83%, 61%, 37% and 63% respectively for the TFF, TFFSW, CT and CFC processes. Through electron microscopy, it was possible to visualize cell integrity after CFC processing, and the major vaccine antigen, pertussis toxin, was detected in the preparation by western blot. As for immunogenicity, anti-pertussis IgG antibodies were detected by ELISA and preliminary results showed no differences in the B. pertussis colonization of lungs in mice immunized with wPlow or wP. We can conclude that the TFF and TFFSW processes were not efficient in removing the endotoxic activity of wPlow, although LPS reduction occurred. Although the processes using industrial centrifuges were efficacious in the removal of LPS and in the reduction of endotoxic activity, there was a low yield in the CT process. The wPlow produced by CFC was immunogenic indicating its potential as a vaccine and that this industrial scale production is a viable process. The characterization of the vaccine strain of Bordetella pertussis by complete genome sequencing was also presented here as part of this work (genbank - number CP010323).

Pertussis (whooping cough) remains an important public health problem in Australia. Although mortality and morbidity from pertussis declined dramatically following the introduction of mass vaccination programs in 1953, the level of morbidity remains unacceptably high for a vaccine-preventable disease. Aims and methods The primary aims of this thesis were (i) to ascertain the epidemiology of pertussis in Australia between 1993 and 2000 by analysing and interpreting sources of routinely collected data on pertussis; and (ii) to examine the effectiveness of vaccination against pertussis in a number of ways. Data from three primary national sources (notifications of disease, hospitalisations for pertussis and death certificates) were used to examine the burden from pertussis in Australia over these eight years. Analyses included the age distribution of cases, temporal and geographic trends, comparisons of notification and hospitalisation data, and the impact of differences in the method of diagnosis of notified cases between years and age groups. In addition to analyses at the national level using data from the national databases, further detailed analyses were undertaken at the State level for New South Wales (NSW), the most populous Australian State. Pertussis vaccine coverage was estimated using data from the recently established Australian Childhood Immunisation Register (ACIR); these data were also used to track the transition from whole-cell to acellular pertussis vaccines. The different types of studies used to evaluate vaccine effectiveness were reviewed, and a method suitable for ongoing estimation of vaccine effectiveness in Australia was developed. This was then applied to the NSW data, to determine the effectiveness of pertussis vaccination in this State. Main findings The annual notification rate for pertussis in Australia ranged from 23�59 per 100 000 population over the eight years. Infants had the highest notification and hospitalisation rates in Australia � they accounted for 5percent of notifications, 61percent of hospitalisations and 100percent of deaths. Age-specific notification and hospitalisation rates in children aged less than two years strongly suggested a protective effect of vaccination, with the greatest reduction in rate coinciding with eligibility to receive a second dose of pertussis vaccine at four months of age. Notification rates among 5�9 year olds progressively decreased in successive age cohorts, consistent with an effect of the introduction in 1994 of a pertussis vaccine booster for preschool-aged children. Although adults (persons aged 15 years or more) accounted for half the notifications, they had the lowest notification rate. The highest numbers of pertussis notifications were in 1997, when most jurisdictions experienced an epidemic. Notification and hospitalisation rates varied across the States and Territories and also across smaller geographic regions in NSW. Areas and years with high notification rates tended to also have high hospitalisation rates, suggesting that trends in notifications reflected trends in incidence. The number of infant hospitalisations in NSW between July 1993 and June 1999 exceeded the number of notifications by 32percent, highlighting the extent of under-notification. Overall, and particularly amongst those aged more than 12 months, the majority of cases notified in NSW were based on the results of serological tests. The proportion diagnosed by culture of the organism was greatest in infants; the proportion diagnosed by serological tests increased with age. There was no evidence that the use of serology had increased since 1994 in NSW, hence changes in notification rates after this time are unlikely to be attributable to increased use of serological diagnosis. ACIR records indicated that in December 2000, 92percent of one-year-old children had received three doses of diphtheria-tetanus-pertussis (DTP) vaccine and 90percent of two-year-olds had received four doses. Vaccine coverage varied by jurisdiction. Since 1997, there was an increased use of DTP vaccines containing acellular pertussis components with a corresponding decrease in the use of vaccines containing whole-cell components. In 2000, almost all DTP vaccines administered contained acellular pertussis components. The results of the vaccine effectiveness study showed that pertussis vaccination was highly effective at preventing pertussis in NSW children, as measured by notified cases. Vaccine effectiveness was highest (91percent) in the youngest age group (8�23 months) and lowest (78percent) in the oldest age group (9�13 years). The screening method has not previously been used to estimate pertussis vaccine effectiveness in Australia. Conclusions This thesis demonstrates the value of integrating varied data sources in estimating the disease burden from pertussis. The data presented here show that the disease burden is substantial in all age groups, despite high levels of vaccine coverage in infants and children. This problem of disease control does not appear to be due to lack of vaccine effectiveness, but there is evidence of waning immunity over time. The analyses presented here form a basis for the ongoing monitoring of trends in pertussis epidemiology following the replacement of whole-cell by acellular pertussis vaccines, and will assist consideration of the need for additional booster doses in adolescents and adults.

Thèse de doctorat : Sciences de la vie et de la santé. Biologie cellulaire et moléculaires des épithéliums : Paris 12 : 2004. / Version électronique uniquement consultable au sein de l'Université Paris 12 (Intranet). Titre provenant de l'écran-titre. Bibliogr. : 332 réf.

Pertussis (whooping cough) remains an important public health problem in Australia. Although mortality and morbidity from pertussis declined dramatically following the introduction of mass vaccination programs in 1953, the level of morbidity remains unacceptably high for a vaccine-preventable disease. Aims and methods The primary aims of this thesis were (i) to ascertain the epidemiology of pertussis in Australia between 1993 and 2000 by analysing and interpreting sources of routinely collected data on pertussis; and (ii) to examine the effectiveness of vaccination against pertussis in a number of ways. Data from three primary national sources (notifications of disease, hospitalisations for pertussis and death certificates) were used to examine the burden from pertussis in Australia over these eight years. Analyses included the age distribution of cases, temporal and geographic trends, comparisons of notification and hospitalisation data, and the impact of differences in the method of diagnosis of notified cases between years and age groups. In addition to analyses at the national level using data from the national databases, further detailed analyses were undertaken at the State level for New South Wales (NSW), the most populous Australian State. Pertussis vaccine coverage was estimated using data from the recently established Australian Childhood Immunisation Register (ACIR); these data were also used to track the transition from whole-cell to acellular pertussis vaccines. The different types of studies used to evaluate vaccine effectiveness were reviewed, and a method suitable for ongoing estimation of vaccine effectiveness in Australia was developed. This was then applied to the NSW data, to determine the effectiveness of pertussis vaccination in this State. Main findings The annual notification rate for pertussis in Australia ranged from 23�59 per 100 000 population over the eight years. Infants had the highest notification and hospitalisation rates in Australia � they accounted for 5percent of notifications, 61percent of hospitalisations and 100percent of deaths. Age-specific notification and hospitalisation rates in children aged less than two years strongly suggested a protective effect of vaccination, with the greatest reduction in rate coinciding with eligibility to receive a second dose of pertussis vaccine at four months of age. Notification rates among 5�9 year olds progressively decreased in successive age cohorts, consistent with an effect of the introduction in 1994 of a pertussis vaccine booster for preschool-aged children. Although adults (persons aged 15 years or more) accounted for half the notifications, they had the lowest notification rate. The highest numbers of pertussis notifications were in 1997, when most jurisdictions experienced an epidemic. Notification and hospitalisation rates varied across the States and Territories and also across smaller geographic regions in NSW. Areas and years with high notification rates tended to also have high hospitalisation rates, suggesting that trends in notifications reflected trends in incidence. The number of infant hospitalisations in NSW between July 1993 and June 1999 exceeded the number of notifications by 32percent, highlighting the extent of under-notification. Overall, and particularly amongst those aged more than 12 months, the majority of cases notified in NSW were based on the results of serological tests. The proportion diagnosed by culture of the organism was greatest in infants; the proportion diagnosed by serological tests increased with age. There was no evidence that the use of serology had increased since 1994 in NSW, hence changes in notification rates after this time are unlikely to be attributable to increased use of serological diagnosis. ACIR records indicated that in December 2000, 92percent of one-year-old children had received three doses of diphtheria-tetanus-pertussis (DTP) vaccine and 90percent of two-year-olds had received four doses. Vaccine coverage varied by jurisdiction. Since 1997, there was an increased use of DTP vaccines containing acellular pertussis components with a corresponding decrease in the use of vaccines containing whole-cell components. In 2000, almost all DTP vaccines administered contained acellular pertussis components. The results of the vaccine effectiveness study showed that pertussis vaccination was highly effective at preventing pertussis in NSW children, as measured by notified cases. Vaccine effectiveness was highest (91percent) in the youngest age group (8�23 months) and lowest (78percent) in the oldest age group (9�13 years). The screening method has not previously been used to estimate pertussis vaccine effectiveness in Australia. Conclusions This thesis demonstrates the value of integrating varied data sources in estimating the disease burden from pertussis. The data presented here show that the disease burden is substantial in all age groups, despite high levels of vaccine coverage in infants and children. This problem of disease control does not appear to be due to lack of vaccine effectiveness, but there is evidence of waning immunity over time. The analyses presented here form a basis for the ongoing monitoring of trends in pertussis epidemiology following the replacement of whole-cell by acellular pertussis vaccines, and will assist consideration of the need for additional booster doses in adolescents and adults.

Thesis (Ph. D.)--University of Virginia, 1997. / Spine title: Characterization of PT receptors. Includes bibliographical references (111-128). Also available online through Digital Dissertations.