Arch Pediatr Infect Dis. 2014 April; 2(2): 196-202. DOI: 10.5812/pedinfect.12505 Research Article Published Online 2014 April 10. Implementation and Assessment of the Use of Real-Time PCR in Routine Diagnosis for Bordetella pertussis Detection in Brazil 1, * 1 1 Daniela Leite , Roberta Morozetti Blanco , Leyva Cecilia Vieira de Melo , Cleiton Eduardo 1 1 1 1 Fiorio , Luciano Moura Martins , Tania Mara Ibelli Vaz , Sueli Aparecida Fernandes , 2 Claudio Tavares Sacchi 1 Center of Bacteriology, National Reference Laboratory for Pertussis, Instituto Adolfo Lutz, Sao Paulo, Brazil 2 Center of Imunology, Instituto Adolfo Lutz, Sao Paulo, Brazil *Corresponding author: Daniela Leite, Center of Bacteriology, National Reference Laboratory for Pertussis, Instituto Adolfo Lutz. Av. Dr. Arnaldo, 351 - 9ºandar, CEP: 01246-902, Sao Paulo-SP, Brazil. Tel: +55-1130682896, Fax: +55-1130819161, E-mail: [email protected]. Received: ; Revised: ; Accepted: May 27, 2013 June 18, 2013 August 27, 2013 Background: Bordetella pertussis is the causative agent of pertussis. In Brazil, laboratory diagnosis of pertussis is based on the culture. In 2010, was standardized the Real-Time PCR TaqMan® in routine diagnosis. Objectives: The aim of this study was to evaluate the impact achieved with the introduction of RT-PCR for the routine diagnosis of pertussis and to compare with the results obtained from culture. Patients and Methods: 4,697 samples of nasopharyngeal secretions collected from suspected pertussis cases and/or contacts were analyzed for RT-PCR and culture, from January 2008 until the end of December 2011. Results: According to the results obtained from the samples 6.9% were culture/RT-PCR positive, 14.8% were positive only for RT-PCR and 0.2% only for culture. Negative samples for both techniques was 3,622 (77.1%) and 1.0% were inconclusive for RT-PCR. Conclusions: The implementation of RT-PCR in routine diagnosis resulted in an increase in laboratory confirmation by almost three times. The RT-PCR assay does not intend to replace the culture technique, but to promote an improvement in the diagnosis of pertussis. Keywords: Bordetella pertussis; Whooping Cough; Diagnosis; Real-Time Polymerase Chain Reaction 1. Background duration of protection following the basic vaccination schedule with a booster dose of wP vaccine or natural Bordetella pertussis, a Gram negative bacterium, is the infection is the same, is estimated to be about 6-12 years. causative agent of whooping cough or pertussis, an in- Some studies show that the duration of protection with fectious respiratory disease of worldwide occurrence the use of aP vaccine is situated within the same time and high prevalence among newborns and children with incomplete immunization. Currently there are eight spe- range (7). cies described in the genus Bordetella including B. pertus- In Brazil, a vaccination program recommended by Min- sis, B. parapertussis, B. holmesii and B. bronchiseptica which istry of Health is three doses with tetravalent vaccine: are the species most commonly associated with respira- diphtheria, wP and tetanus (DTP) + Hib (Haemophilus in- tory infections in humans, although the last one rarely fluenza B) at ages 2, 4 and 6 months age and two boosters infects healthy humans (1-4). of DTP, the first at 15-18 months and the second between Despite mass vaccination programs and good cover- ages 4 and 6 years (8). age in many countries, B. pertussis continues to circulate From 1980 to 1983 more than 40,000 cases of pertussis worldwide and periodically cause epidemics every 3-5 (incidence rate > 30/100,000) were reported in Brazil, but years. Currently, a large number of cases have been re- cases decreased substantially after 1983. In 1990, 15,329 ported among adolescents and adults, to be important cases (incidence rate 10.64/100,000) were reported, the sources of infection for infants and children (5, 6). highest rate observed in the decade. In 1995, 3,798 cases High levels of efficiency have been obtained from both (incidence rate 2.44/100,000) were reported and, there- whole-cell (wP) or acellular (aP) pertussis vaccines. The after, the annual number of cases did not exceed 2,000 Implication for health policy/practice/research/medical education: This paper evaluates the impact achieved with the introduction of RT-PCR for the routine diagnosis of pertussis in Brazil and compares with the results obtained from culture, the gold standard method for diagnosis. Copyright © 2014, Pediartric Infections Research Center. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is prop- erly cited. Leite D et al. (incidence rate 1/100,000). The years 2008 and 2011 re- 3.2. Clinical Samples and Culture ported 1,427 and 2,257 cases (incidence rate 0.71/100,000 We analyzed 4,697 samples of nasopharyngeal secretions and 1.2/100,000) respectively, being considered epidem- collected from patients and/or contacts with suspected ics years by epidemiological surveillance system of the pertussis, assisted in the basic health units distributed in country (9). all Sao Paulo state, from the beginning of January 2008 Estimates from the World Health Organization (WHO) until the end of December 2011. Patients and/or contacts suggest that in 2008, about 16 million cases of pertussis were separated into six age-groups: Group 1: ≤ 2 months occurred worldwide, of which 95% were in developing (n = 1,341); Group 2: 3-6 months (n = 861); Group 3: 7 months countries, where 195,000 children died (10). - 1 year (n = 368); Group 4: 2-9 years (n = 453); Group 5: 10-19 The culture is considered the "gold standard" for detec- years (n = 409) and Group 6: ≥ 20 years (n = 1,034). The age tion of B. pertussis, it is a highly specific method, but with group ranged from eight days old to 85 years old and in 231 a variable sensitivity and lengthy incubation periods. patients and/or contacts was not known. When the material collection is performed at symptom Were considered suspected of pertussis and reported onset (3-4 weeks), high rates of isolation of B. pertussis is to the surveillance system disease: any person who, achieved, but the positivity rate of culture decreases to without other apparent cause, regardless of vaccina- about 15-20% with nasopharyngeal secretion collected six tion status or age, presented symptoms of infection of weeks after the onset of symptoms (11-13). the upper airways with cough lasting at least two weeks Several studies have shown the usefulness of real time and with at least one of the following symptoms: (i) par- polymerase chain reaction (RT-PCR) to detect pathogens. oxysms of coughing, (ii) inspiratory whooping and (iii) The high sensitivity (70-90%) and specificity (86-100%), post-tussive vomiting (i.e. vomiting immediately after along with a shorter response time to results, low risk of coughing) (18). contamination and ease of performance, makes RT-PCR Clinical samples were collected with ultrathin and flex- an alternative method for the diagnosis of many diseas- ible sterile swabs, and immediately placed in a tube with es, among them, the pertussis (13-15). transport Regan-Lowe (RL) medium containing charcoal Traditionally, in Brazil, pertussis laboratory diagnosis agar (Oxoid) semi-solid with 10% sterile defibrinated has been based solely on culture method. Due to the in- sheep blood, and Cephalexin (40 μg/mL). The center of crease in number of notifications of pertussis observed bacteriology, Instituto Adolfo Lutz center and the twelve in other countries (16, 17) and also in Brazil, for over a de- centers of regional laboratories of Instituto Adolfo Lutz cade, there was a need to implement a diagnostic meth- (CLRs - IAL) received and processed the swabs collected at od with higher sensitivity and specificity, with a shorter basic health units according to their areas of coverage. time to release results. Then, in 2010, we standardized the The specimens were cultured on the collection day on Real-Time PCR TaqMan® (RT-PCR) in routine diagnosis of charcoal agar plates (Oxoid) with sheep blood and Ceph- Instituto Adolfo Lutz, the national reference center for alexin incubated at 35-37° C under high humidity ambi- pertussis in Brazil. ent air for twelve days. The samples that showed growth from the third or fourth day, with colonies suggestive 2. Objectives of belonging to the genus Bordetella were confirmed by The aim of this study was to evaluate the impact Gram staining. The biochemical and antigenic character- achieved with the introduction of such technology for izations were realized as described previously (19). the routine diagnosis of pertussis in our settings and to After the culture procedure, the swabs were transferred compare with the results obtained from culture. to dried sterile tubes and kept at -20° C for subsequent phase of extraction, purification and concentration of 3. Patients and Methods bacterial DNA. The swabs processed for culture in CLRs were sent to IAL Central for RT-PCR performing. 3.1. Microorganisms Strains 3.3. Extraction and Purification of Bacterial DNA To evaluate the sensitivity and specificity of RT-PCR, 106 The swabs were thawed, removed from the dry tube B. pertussis strains and a total of 293 strains of microorgan- and stirred with rotational movements into a 1.5 mL mi- isms were analyzed, to several genera and species, which cro tube with 200 µL of free DNA ultrapure sterile water may be present in the human nasopharynx (Table 1). (Roche Diagnostics, Indianapolis, Ind.). From this step, The bacterial strains used in this study belong to the col- the extraction was carried out using QIAampDNA Mini lection of bacteriology research center, Instituto Adolfo Kit (QIAGEN, Valencia, CA) according to the protocol de- Lutz (IAL).