Recent Trends of Antigenic Variation in Bordetella pertussis Isolates in Korea

Pertussis is a representative vaccine-preventable disease. However, there have been recent outbreaks in countries where even higher vaccination against the disease. One reason is the emergence of antigenic variants, which are different to vaccine type. In Korea, reported cases have rapidly increased since 2009. Therefore, we analyzed genotype of strains isolated in 2011-2012 by multilocus sequence typing method. As expected, the genotype profiles of tested genes dramatically changed. The major sequence type changed from ST1 to ST2, and new sequence type (ST8) appeared. In the minimum spanning tree, recent isolates belonging to the ACC-I-ST3 subgroup were detected that were composed of ST2, ST3, and ST6. In particular, the ST2 frequency increased to 81%. The novel ST8 was linked to the increased frequency of ST2. In addition, toxic strains carrying the ptxP3 promoter type were confirmed. This ptxP3 type emerged from 2009 and its frequency had increased to 100% in 2012. Based on these results, it can be inferred that the genotypic changes in the currently circulating strains are strongly associated with the recent increasing of pertussis in Korea. Therefore, the surveillance system should be strengthened, and genetic characterization of the isolates should be expanded to the whole genome sequence level.

A Multicenter Study of Pertussis Infection in Adults with Coughing in Korea: PCR-Based Study / 결핵및호흡기질환

BACKGROUND: Limited data on the incidence and clinical characteristics of adult pertussis infections are available in Korea. METHODS: Thirty-one hospitals and the Korean Centers for Disease Control and Prevention collaborated to investigate the incidence and clinical characteristics of pertussis infections among adults with a bothersome cough in non-outbreak, ordinary outpatient settings. Nasopharyngeal aspirates or nasopharyngeal swabs were collected for polymerase chain reaction (PCR) and culture tests. RESULTS: The study enrolled 934 patients between September 2009 and April 2011. Five patients were diagnosed as confirmed cases, satisfying both clinical and laboratory criteria (five positive PCR and one concurrent positive culture). Among 607 patients with cough duration of at least 2 weeks, 504 satisfied the clinical criteria of the US Centers for Disease Control and Prevention (i.e., probable case). The clinical pertussis cases (i.e., both probable and confirmed cases) had a wide age distribution (45.7+/-15.5 years) and cough duration (median, 30 days; interquartile range, 18.0~50.0 days). In addition, sputum, rhinorrhea, and myalgia were less common and dyspnea was more common in the clinical cases, compared to the others (p=0.037, p=0.006, p=0.005, and p=0.030, respectively). CONCLUSION: The positive rate of pertussis infection may be low in non-outbreak, ordinary clinical settings if a PCR-based method is used. However, further prospective, well-designed, multicenter studies are needed.

The Clinical Features of Pertussis in Infancy / 소아감염

PURPOSE: Pertussis was very common in the past, but reported cases have dramatically decreased. The improvement of vaccination programs and unreadiness of laboratory confirmation seems to have developed this situation. This study investigated the frequency of pertussis among infants with a paroxysmal cough and compared the clinical characteristics between infants with and without pertussis. METHODS: Between June and November 2006, 27 infants admitted to the hospital that were 15-90 days old with a history of a cough for more than seven days were enrolled. The cough was described as: paroxysmal, whooping, and post-tussive vomiting. PCR and cultures for Bordetella pertussis with nasopharyngeal aspirates were obtained. The patients were divided into two groups: (1) the pertussis group that had positive results by PCR or culture; (2) the control group that had negative results by PCR and culture. Clinical and laboratory characteristics were compared between the two groups. RESULTS: Among the 27 cases, five (18.5%) were finally diagnosed with pertussis. Only one out of the five pertussis cases was initially diagnosed with a pertussis-like syndrome on admission. Compared to the group without pertussis, the pertussis group had a significantly higher frequency of: no fever (P=0.043), a paroxysmal cough (P=0.040), cyanosis (P=0.001), non-immunized status for DTaP (P=0.047), normal auscultation (P=0.028), normal chest X-ray findings (P=0.027), high absolute lymphocyte count (P=0.039), and low CRP (P=0.046). The patients with the diagnosis of pertussis had a significantly longer duration of coughing (27.2+/-10.6 vs. 12.6+/-5.6 days, P=0.039). CONCLUSION: Pertussis should be suspected in any infant with typical symptoms of pertussis in addition to: a persistent cough without fever, accompanied by paroxysms or cyanosis prior to the age of DTaP immunization. Active laboratory confirmation should be carried out to confirm more cases with pertussis.

Discriminative PCR of Bordetella pertussis from closely related Bordetella species using 16S rDNA Gene / 감염과화학요법

BACKGROUND: Polymerase-chain reaction (PCR) detection is useful to diagnosis of pertussis at initial stage because the growth rate of Bordetella pertussis (B. pertussis) is relatively slow. Currently, the primer set for the insertion sequence IS481 (BP primer) is used widely for PCR detection of B. pertussis. However, the cross-reactivity of BP primer set with Bordetella holmesii (B. holmesii) was reported recently. Therefore, discrimination of B. pertussis and B. holmesii is needed in PCR step. For this reason, we developed new primer sets based on 16S rDNA sequence for diagnostic use and estimated the efficiency of these new primer sets. MATERIALS AND METHODS: The specific PCR primers were designed from the aligned sequence matrix of 16S rDNA genes of various Bordetella species. The specificity of designed primers were estimated using clinically important 4 Bordetella species, B. pertussis, B. holmesii, Bordetella parapertussis (B. parapertussis) and Bordetella bronchiseptica (B. bronchiseptica). The sensitivity to B. pertussis of designed primers was also estimated and compared with BP primer set. RESULTS: As the results, the developed new primer set successfully distinguished B. pertussis and other Bordetella species containing B. holmesii. In the sensitivity assay, the detectable limits of 16S-F2/16S-R1 primer set for B. pertussis were revealed as 5 pg of genomic DNA and 105 cells/mL of cell suspension. In addition to these, identical results between BP with primer and new primer were obtained in clinical samples. CONCLUSION: In this study, the specific primer set for B. pertussis was developed based on 16S rDNA sequence and this primer set did not show cross-reactivity to B. holmesii. In addition to these, the applicability of this primer set to the clinical specimens was also confirmed.

Discriminative PCR of Bordetella pertussis from closely related Bordetella species using 16S rDNA Gene / 감염과화학요법

BACKGROUND: Polymerase-chain reaction (PCR) detection is useful to diagnosis of pertussis at initial stage because the growth rate of Bordetella pertussis (B. pertussis) is relatively slow. Currently, the primer set for the insertion sequence IS481 (BP primer) is used widely for PCR detection of B. pertussis. However, the cross-reactivity of BP primer set with Bordetella holmesii (B. holmesii) was reported recently. Therefore, discrimination of B. pertussis and B. holmesii is needed in PCR step. For this reason, we developed new primer sets based on 16S rDNA sequence for diagnostic use and estimated the efficiency of these new primer sets. MATERIALS AND METHODS: The specific PCR primers were designed from the aligned sequence matrix of 16S rDNA genes of various Bordetella species. The specificity of designed primers were estimated using clinically important 4 Bordetella species, B. pertussis, B. holmesii, Bordetella parapertussis (B. parapertussis) and Bordetella bronchiseptica (B. bronchiseptica). The sensitivity to B. pertussis of designed primers was also estimated and compared with BP primer set. RESULTS: As the results, the developed new primer set successfully distinguished B. pertussis and other Bordetella species containing B. holmesii. In the sensitivity assay, the detectable limits of 16S-F2/16S-R1 primer set for B. pertussis were revealed as 5 pg of genomic DNA and 105 cells/mL of cell suspension. In addition to these, identical results between BP with primer and new primer were obtained in clinical samples. CONCLUSION: In this study, the specific primer set for B. pertussis was developed based on 16S rDNA sequence and this primer set did not show cross-reactivity to B. holmesii. In addition to these, the applicability of this primer set to the clinical specimens was also confirmed.

Genotypic and Phenotypic Analysis Among Clinical Isolate of Streptococcus pyogenes in Seoul , Korea

A total of 152 strains of Streptococcus pyogenes were isolated from patients with pharyngitis, scarlet fever, skin infection, or invasive streptococcal infections in Seoul, Korea from January 1988 to December 1999. All isolates were epidemiologically characterized to decide phenotypes by T protein serotype and serum opacity factor (OF) detection. Genetic diversity of the isolates were analyzed by emm genotyping and pulsed-field gel electrophoresis (PFGE). T protein serotype showed 17 kinds in distribution and T12 (40.1% of study strains), T4 (19.1%), and T1 (7.9%) were the prevalent ones. When sources of S. pyogenes isolates were analyzed by T serotype distribution, T12 type was predominant in pharyngitis and skin infection isolates which contributed to 30 strains (49.2%) and 11 strains (18.0%), respectively. When T serotype of S. pyogenes isolates were analyzed by emm genotype distribution, of the 61 isolates of T12 type, 48 strains (78.7%) belonged to the emm type 12 (M12) and of the 29 isolates of T4 type, 27 strains (93.1%) belonged to the emm genotype 4 (M4). PFGE of genomic DNA of different emm genotype (emm12, emm4 and emm1) showed distinctive patterns. When the DNA of same emm gene type isolates were analyzed genetic relatedness by PFGE pattern, emm4, emm1, and emm12 types showed over 90%, 75%, and 70% of genetic similarity, respectively. Therefore, it was suggested that these emm genotype isolates were closely related genetically whereas among the isolates of other emm genotypes showed less than 30% of genetic similarity. Show genotypes are more diverse in comparison with phenotypes. In even epidemiologically unrealated isolates, genetic subtypes appeared correlated. The phenotypic and genotypic analysis used in the study were discriminative and appropriate for epidemiological study of S. pyogenes.

Pulsed field gel electrophoresis profile of erythromycin-clindamycin resistant Streptococcus pyogenes isolated in Korea

Ninety two strains of Streptococcus pyogenes were isolated from patients with pharyngitis, scarlet fever, skin infection, and invasive streptococcal infections in Seoul, Korea from January to December, 1998. All isolates were epidemiologically characterized by T protein serotype, and serum opacity factor (OF) detection to phenotypes. To analyze the genetic relationship, fifty two isolates including 32 erythromycin-clindamycin (Em-Cm) resistant strains, 20 antimicrobial susceptible strains were attempted to the pulsed-field gel electrophoresis (PFGE). T protein serotype showed 16 kinds in distribution including T12 and T4. Among the total isolates, 40 strains (43.5%) belonged to the T12 serotype and twenty strains (21.7%) to T4 serotype. On the other hand, when infection aspect of S. pyogenes isolates were analysed by T serotype distribution, T12 type was predominant for pharyngitidis which contributed to 21 strains (53%) and for skin infection isolates which contributed to 11 strains (28%), respectively. In case of T4 type, it was the most predominant pharyngitidis isolates which contributed to 8 strains (40%). In T serotype distribution of Em-Cm resistant strains, 27 strains (84%) of the thirty two showed T12 serotype. In minimum inhibitory concentration (MIC) values of Em-Cm resistance isolates, thirty two isolates showed resistant to erythromycin 27 strains (84%), had high MIC of >128 mug/ml. And also to clindamycin, twenty two strains (69%) had high MIC of >128 mug/ml. When OF detection of Em-Cm resistance of S. pyogenes isolates were analyzed by T serotype distribution, T12 serotype isolates revealed that all of the isolates except one strain were OF negative. In PFGE profile analysis to Em-Cm resistance isolates, of the twenty seven, Em-Cm resistance of T12 serotype isolates, 26 strains showed identical PFGE profile and all of these isolates revealed that OF negative. Eighty four percent of Em-Cm resistance S. pyogenes isolates had identical phenotype and PFGE profile. These results strongly suggested that the Em-Cm resistant S. pyogenes isolates from Seoul area showed close genetic correlation and PFGE could be available tool for molecular epidemiology.