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Molecular epidemiology and antimicrobial susceptibility of outbreak-associated Corynebacterium diphtheriae in Thailand, 2012
Infection, Genetics and Evolution 75 (2019) 104007
Contents lists available at ScienceDirect
Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
Research paper
Molecular epidemiology and antimicrobial susceptibility of outbreakassociated Corynebacterium diphtheriae in Thailand, 2012
T
Wantana Paveenkittiporna, Saowalak Sripakdeea, Onchuda Koobkratoka, Somchai Sangkitporna, ⁎ Anusak Kerdsinb, a b
National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Nonthaburi, Thailand Faculty of Public Health, Kasetsart University Chalermphrakiat Sakon Nakhon Province Campus, Sakon Nakhon, Thailand
A R T I C LE I N FO
A B S T R A C T
Keywords: Antimicrobial susceptibility Corynebacterium diphtheriae MLST Outbreak Thailand
Infections caused by Corynebacterium diphtheriae remain endemic in many countries. Since the implementation of the DTP (Diphtheria-Tetanus-Pertussis) vaccination program in 1977, only sporadic diphtheria cases have been reported in Thailand. In 2012, a diphtheria outbreak occurred in rural Thailand and 38 cases were reported, with the majority being adults (mean 22.1 years, range 5–72 years). The current study determined the genetic diversity of C. diphtheriae isolated from 83 individuals associated with either sporadic (n = 34) from 1994, 1996, 1997, 1998, 1999, 2000, 2012, and 2018, or 2012 outbreak (n = 49) diphtheria occurrences in Thailand. Antimicrobial susceptibility testing was performed on 41/83 isolates using broth microdilution. All sporadic (n = 27) and epidemic (n = 14) C. diphtheriae isolates (41/41; 100%) were susceptible to erythromycin (≤0.5 μg/ml), clindamycin (≤0.5 μg/ml), gentamicin (≤ 4 μg/ml), ciprofloxacin (≤1 μg/ml), and vancomycin (2 μg/ml), except tetracycline with a resistance rate of 34.1% (14/41 isolates). All isolates were intermediately resistant to penicillin (MIC range, 0.25–2 μg/ml). Multilocus sequence typing (MLST) revealed 17 sequence types (STs) among 83C. diphtheriae isolates. For the 2012 outbreak isolates, the predominant ST was ST243 (n = 34/ 49; 69.4%), followed by ST245 (n = 5/49; 10.2%) and ST244 (n = 4/49; 8.1%), whereas the main STs among the sporadic isolates were ST248 (n = 15/34; 44.1%), followed by ST209 (n = 7/34; 20.6%) and ST258 (n = 3/ 34; 8.8%). The ST243 outbreak strain was a single-locus variant of sporadic ST258. Phylogenetic analysis using concatenated sequences of 7 MLST genes from 17 STs revealed that ST243, ST248, and ST258 were located in the same cluster and ST243 appeared to have evolved from ST258, an endemic strain. This study highlights the importance of epidemiological surveillance together with characterization of C. diphtheriae strains to help inform the future control and prevention of diphtheria.
1. Introduction Diphtheria is a longstanding disease caused by toxigenic strains of Gram-positive Corynebacterium diphtheriae and the introduction of the diphtheria toxoid vaccine in 1923 and widespread mass immunization in the 1940s and 1950s led to the near elimination of the disease in the industrialized world (Efstratiou and George, 1996). Nonetheless, diphtheria remains endemic and has re-emerged in many developing countries around the globe, such as in Eastern Europe, Southeast Asia, South America, and the Indian subcontinent (Mattos-Guaraldi et al., 2003). Thailand's National Expanded Programme on Immunization (EPI), focusing on diphtheria, pertussis, tetanus, poliomyelitis, measles and tuberculosis, was initiated in 1977. By 2018, diphtheria, tetanus,
⁎
pertussis (DTP) vaccine coverage in Thailand was estimated to be 97% among infants below one year of age (World Health Organization, 2019). The DTP vaccine coverage in migrant school children on the Thailand-Myanmar border had the poorest coverage among children < 7 years of age with 89.2, 76.2, and 63.8% for the first, second, and third doses, respectively (Kaji et al., 2016). Booster of tetanus and diphtheria containing vaccine (Td) among age group ≥7 years showing 97.8%, 91.9%, 60.7% coverage for Td1, Td2 and Td3 vaccination, respectively (Kaji et al., 2016). According to a World Health Organization report, the numbers of cases for Thailand in 1996, 1997, 1998, 1999, and 2000 were 53, 38, 43, 52, and 15, respectively (World Health Organization, 2018). In June 2012 a diphtheria outbreak occurred in Loei province, northeast Thailand resulting in 38 cases and 2 cases were died. The first
Corresponding author at: Faculty of Public Health, Kasetsart University Chalermphrakiat Sakon Nakhon Province Campus, Sakon Nakhon 47000, Thailand. E-mail address: [email protected] (A. Kerdsin).
https://doi.org/10.1016/j.meegid.2019.104007 Received 4 May 2019; Received in revised form 12 August 2019; Accepted 15 August 2019 1567-1348/ © 2019 Elsevier B.V. All rights reserved.
Infection, Genetics and Evolution 75 (2019) 104007
W. Paveenkittiporn, et al.
tetracycline were determined among 41 out of the 83 isolates (sporadic = 27; outbreak = 14) using broth microdilution and results were interpreted according to Clinical Laboratory Standards Institute guidelines (CLSI, 2015). These MIC tested isolates were selected as representative STs that cover 17 STs distribution in the study.
case was a 40-year-old immunocompromised alcoholic male, who subsequently died of acute myocarditis. The second case was a 25-yearold male from a nearby village; subsequent cases were reported in a village close to where the second case lived and then spread to neighboring provinces. The Ministry of Public Health stated that during June 2012–January 2013, 38 laboratory-confirmed cases, 5 probable cases, and 93 asymptomatic carriers were reported from northeastern Thailand and the majority of confirmed cases in the 2012 outbreak were reported among adults (Wanlapakorn et al., 2014). This study determined the genetic relationship and antimicrobial susceptibility profiles among C. diphtheriae isolates from the 2012 outbreak, and from sporadic cases during 1994–2018 in Thailand.
2.3. Molecular characterization A multiplex PCR assay was performed for confirmation of C. diphtheriae based on dtxR (a species-specific marker), and for detection of strains carrying toxA and toxB (Pimenta et al., 2008). Multilocus sequence typing (MLST) was performed as described elsewhere (Bolt et al., 2010). MLST alleles and the resulting STs were assigned using the C. diphtheriae MLST database (https://pubmlst.org/cdiphtheriae/). eBURST was used to identify clonal complexes (CC) for these C. diphtheriae isolates and to compare ST relatedness to STs of other isolates currently available in the MLST database (Feil et al., 2004). Construction of phylogenetic trees for all STs in this study was performed via Phylogeny.fr (Dereeper et al., 2008).
2. Materials and methods 2.1. Isolates A total of 83 C. diphtheriae clinical isolates were characterized, with 49 (designated the outbreak group) isolated during the outbreak period in 2012 and 34 (designated the sporadic group) from sporadic cases that occurred throughout the country, from 1994 (n = 1), 1996 (n = 1), 1997 (n = 2), 1998 (n = 1), 1999 (n = 3), 2000 (n = 1), 2012 (n = 21; different region of outbreak), and 2018 (n = 4) (Table 1).
3. Results In total, identification of 83 C. diphtheriae strains was confirmed using conventional biochemical tests and PCR as described above. We characterized two groups of C. diphtheriae isolates as either sporadic or belonging to the 2012 outbreak. The sporadic group (n = 34) was composed of 30 toxigenic and 4 non-toxigenic isolates while the 2012 outbreak group (n = 49) from Loei and nearby provinces including Nong Bua Lamphu, Phetchabun, and Udon Thani consisted of 37 toxigenic and 12 non-toxigenic isolates based on Elek's test. Therefore, in total, there were 67 toxigenic and 16 non-toxigenic isolates and all were concordant with the PCR assay results for toxA and toxB as described above (Table 1). Based on the Clinical Laboratory Standards Institute guidelines (CLSI M45, 2015), antimicrobial susceptibility testing revealed that both sporadic and outbreak C. diphtheriae including toxigenic and nontoxigenic isolates (n = 41) were susceptible to erythromycin, clindamycin, vancomycin, gentamicin, and ciprofloxacin used in this study, except tetracycline which had a resistance rate of 34.1% (Table 2). The majority of outbreak isolates (11/14; 78.6%) were resistant to tetracycline whereas 11.1% of the sporadic isolates (3/27) were resistant to tetracycline (Table 2). Susceptibility testing of all isolates were intermediately resistant to penicillin (MIC range 0.25–0.5 μg/ml). MLST revealed 17 STs in the C. diphtheriae isolates. The predominant STs among the 2012 outbreak isolates were ST243 (n = 34; 69.4%), followed by ST245 (n = 5; 10.2%) and ST244 (n = 4; 8.1%) (Table 1, Fig. 1), whereas the predominant STs in the sporadic isolates were ST248 (n = 15; 44.1%), followed by ST209 (n = 7; 20.6%) and ST258 (n = 3; 8.8%) (Table 1, Fig. 1). All of the ST243 and ST209 isolates were toxin-producing whilst 86.6% (13/15) of sporadic ST248 isolates were toxin-producing (Table 1). The eBURST analysis showed that outbreak-associated ST243 (allelic profile: 2,4,8,19,7,3,9) is a single-locus variant of sporadic ST258 (allelic profile: 2,4,8,1,7,3,9). ST246 (allelic profile: 2,1,45,46,5,16,9) and ST209 (allelic profile: 3,3,4,2,3,3,3) are single-locus variants of ST254 (allelic profile: 2,1,71,46,5,16,9) and ST255 (allelic profile: 3,3,4,47,3,3,3), respectively (Figs. 1 and 2). The phylogenetic tree constructed using concatenated sequences of 7 MLST genes from the 17 STs revealed that ST248, ST258, and ST243 were located in the same cluster and that ST243 and ST258 appear to have evolved from ST248, an endemic strain found throughout Thailand (Fig. 2).
2.2. Microbiological methods Conventional biochemical tests were used to confirm C. diphtheriae identification (Thompson et al., 1983). Briefly, after culturing on sheep blood agar for 18–24 h at 37 °C, the organisms were tested using Gram stain, catalase production, reduction of nitrate, urea hydrolysis, starch hydrolysis, and production of acid in glucose, maltose, and sucrose (Thompson et al., 1983). Elek's test was conducted to detect the exotoxin produced by clinical isolates (Efstratiou and Maple, 1994). The minimum inhibitory concentrations (MIC) of penicillin, erythromycin, vancomycin, clindamycin, gentamicin, ciprofloxacin, and Table 1 Characteristics of C. diphtheriae from Thailand (n = 83). Category
Sequence type
Year of isolation
Toxin production
No. of isolates
Outbreak
123 209 243 244
2012 2012 2012 2012
245
2012
246 247 259
2012 2012 2012
Negative Positive Positive Positive Negative Positive Negative Negative Negative Negative
248
2012
2 1 34 1 3 1 4 1 1 1 49 9 2 4 1 1 1 4 2 1 1 1 1 1 1 1 1 1 1 34
Total Sporadic
209
255 258
243 249 253 254 256 257 285 Total
2018 1998 1999 2000 2012 2012 1994 1996 1997 1997 2012 2012 2012 1999 1999 2012
Positive Negative Positive Positive
Positive Positive
Positive Positive Negative Negative Positive Positive Positive
4. Discussion The genetic diversity and antimicrobial susceptibility among C. diphtheriae isolates associated with the 2012 outbreak and from 2
Infection, Genetics and Evolution 75 (2019) 104007
W. Paveenkittiporn, et al.
Table 2 Antimicrobial susceptibility of C. diphtheriae (n = 41) determined by broth microdilution. Categories
Outbreak
Total Sporadic
Sequence types
Toxin production
243 244 245 123 209 246 247 259
Positive Positive Positive Negative Positive Negative Negative Negative
248
Positive Negative Positive Positive Positive Positive Positive Negative Negative Positive Positive Positive
209 255 258 243 249 253 254 256 257 285 Total
No. of isolates
7 1 1 1 1 1 1 1 14 8 2 5 2 3 1 1 1 1 1 1 1 27
Antimicrobial susceptibility testing Penicillin
Clindamycin
Vancomycin
Tetracycline
Erythromycin
Gentamicin
Ciprofloxacin
I I I I I I I I
S S S S S S S S
S S S S S S S S
R R R R S S R S
S S S S S S S S
S S S S S S S S
S S S S S S S S
I I I I I I I I I I I I
S S S S S S S S S S S S
S S S S S S S S S S S S
S S S S S S R R S S R S
S S S S S S S S S S S S
S S S S S S S S S S S S
S S S S S S S S S S S S
S = Susceptible; I = Intermediate; R = Resistant.
tetracycline has been reported in C. diphtheriae isolates from Brazil (12.8%) and Algeria (71.3%) (Pereira et al., 2008; Benamrouche et al., 2016). Susceptibility to penicillin, the first drug of choice, in all isolates in this study had not changed over time based on the intermediate level. Benamrouche et al. (2016) reported that 57.3% of 157 C. diphtheriae isolates from Algeria between 1992 and 2015 were intermediate for penicillin G. In Brazil, 14.8% of the 47 C. diphtheriae isolates were resistant to penicillin G (Pereira et al., 2008). The majority (7/11) C. diptheriae strains from Italy were intermediately resistant to penicillin (Hunolstein et al., 2003). Accordingly, our findings and previous
sporadic cases during 1994–2012 and 2018 were determined in this study. The outbreak was predominantly due to ST243 whereas the main STs among the sporadic isolates were ST248, however, both STs are closely related. Both toxigenic and non-toxigenic C. diphtheriae isolates in this study were susceptible to almost all antibiotics, except tetracycline and penicillin that showed reduced susceptibility. There were no differences in the antibiotic susceptibility patterns between the toxigenic and non-toxigenic isolates. Resistance to tetracycline was higher among the 2012 outbreak isolates compared to the sporadic isolates. Reduced susceptibility to
Fig. 1. eBURST analysis of sequence types (n = 623 STs) submitted to the C. diphtheriae MLST database (accessed on Apr 15, 2019). Outbreak and sporadic isolate sequence types from Thailand are shown as red and green dots, respectively. Clonal complexes and predicted founding STs are indicated by blue dots. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 3
Infection, Genetics and Evolution 75 (2019) 104007
W. Paveenkittiporn, et al.
Fig. 2. Unrooted tree based on the alignments of concatenated sequences in 17 sequence types of C. diphtheriae from Thailand using the neighbour-joining method. Scale bar indicates sequence dissimilarity.
not able to compare the characteristics of the outbreak strains between Lao PDR and Thailand, although the outbreak occurred in the same period. Sornbundit et al. (2017) simulated a mathematical model to explain diphtheria transmission dynamics in Thailand (Sornbundit et al., 2017). Their simulations predicted outbreaks in the future could occur in approximately 2020 and 2045 (Sornbundit et al., 2017). The World Health Organization recommendation for diphtheria booster frequency is every 10 years after completing the primary dose (Scheifele and Ochnio, 2009). Although the rate of diphtheria toxin antibody loss was estimated to be 1/(15 × 365) day−1 (Hammarlund et al., 2016) and the overall seroprotection rate of diphtheria among Thai adults was 87.7–90.9% (Wanlapakorn et al., 2014), a booster vaccination every 10 years is recommended to maintain high protective vaccine-induced antibody titers for prevention of diphtheria in Thailand. This is essential as the number of susceptible hosts (especially in adults) is increasing which can lead to greater immunization waning. Moreover, Thailand's neighboring countries (Lao PDR, Cambodia, Myanmar) still have high incidences of diphtheria, which could spread across border. In addition, circulation of C. diphtheriae strains should be monitored for epidemiological control and prevention.
reports suggested that treatment with penicillin may not be effective; therefore, treatment with macrolides should rather be recommended in these regions. Among the STs in the outbreak and sporadic groups suggested that the 2012 outbreak C. diphtheriae isolates might be silently circulating in the area undetected (asymptomatic colonization) for a long time before an outbreak occurs. Several STs (ST123, ST243, ST244, ST245, ST246, ST247) were found in the same outbreak period. Among the outbreak strains, ST123 and ST245 were most closely related with 5 identical alleles (atpA, dnaE, fusA, odhA, rpoB) of the 7 MLST genes, whereas the remaining outbreak-associated STs were unrelated. ST243 (toxigenic isolates) were likely the first generation due to it being found throughout the epidemic in the outbreak area. This ST may possess better adaptive properties and abilities to disseminate in current populations. In addition, the majority of the cases were adults, so that waning vaccine-induced immunity may accelerate the infections (Wanlapakorn et al., 2014). However, it was not clear how ST243 emerged because almost all sporadic STs were different from the outbreak STs, with the exception of the ST243 (toxin-producing) strain which was an isolate from a sporadic event occurring 15 years ago (1997) in Chaiyabhum province (Table 1). This finding indicated that ST243 was present in Thailand before the outbreak in 2012. The MLST database revealed that ST243 has subsequently also been isolated in the UK in 2013 [https://pubmlst. org/cdiphtheriae/]. Phylogenetic analysis suggested that ST243 appeared to have evolved from ST258. Additionally, it should be noted that ST248 is an endemic strain in Thailand as this ST was identified throughout the country before and after the 2012 outbreak. However, ST248 has been found in Malaysia, the Philippines, and Germany (Khalid et al., 2019; https://pubmlst.org/cdiphtheriae/). During April 1, 2012–May 31, 2013, a diphtheria outbreak was reported in neighboring Lao PDR (Sein et al., 2016). However, of the 168 suspected cases reported from 7 of 17 provinces, 62 matched the clinical case definition and only 4 out of 21 cases were culture positive for toxigenic C. diphtheriae (Sein et al., 2016). Unfortunately, there was no report on microbiological or MLST characterization of the outbreak C. diphtheriae isolates in Lao PDR (Sein et al., 2016). Therefore, we were
Funding This study was supported by a grant from the Department of Medical Sciences, Ministry of Public Health.
Declaration of Competing Interest None.
Acknowledgement We thank the Kasetsart University Research and Development Institute (KURDI), Bangkok, Thailand for English editing assistance. 4
Infection, Genetics and Evolution 75 (2019) 104007
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Benamrouche, N., Hasnaoui, S., Badell, E., Guettou, B., Lazri, M., Guiso, N., Rahal, K., 2016. Microbiological and molecular characterization of Corynebacterium diphtheriae isolated in Algeria between 1992 and 2015. Clin. Microbiol. Infect. 22, 1005.e1–1005.e7. Bolt, F., Cassiday, P., Tondella, M.L., Dezoysa, A., Efstratiou, A., Sing, A., Zasada, A., Bernard, K., Guiso, N., Badell, E., Rosso, M.L., Baldwin, A., Dowson, C., 2010. Multilocus sequence typing identifies evidence for recombination and two distinct lineages of Corynebacterium diphtheriae. J. Clin. Microbiol. 48, 4177–4185. Clinical and Laboratory Standard Institute, 2015. Method for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria, 3rd ed. M45. Dereeper, A., Guignon, V., Blanc, G., Audic, S., Buffet, S., Chevenet, F., Dufayard, J.F., Guindon, S., Lefort, V., Lescot, M., Claverie, J.M., Gascuel, O., 2008. Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Res. 36, W465–W469. https://doi.org/10.1093/nar/gkn180. (Web Server issue). Efstratiou, A., George, R.C., 1996. Microbiology and epidemiology of diphtheria. Rev. Med. Microbiol. 7, 31–42. Efstratiou, A., Maple, P.A.C., 1994. Manual for the laboratory diagnosis of diphtheria. In: ICP/EPI038 (C). Expanded Programme on Immunisation in the European Region of WHO, Copenhagen, Denmark. Feil, E.J., Li, B.C., Aanensen, D.M., Hanage, W.P., Spratt, B.G., 2004. eBURST: inferring patterns of evolutionary descent among clusters of related bacterial genotypes from multilocus sequence typing data. J. Bacteriol. 86, 1518–1530. Hammarlund, E., Thomas, A., Poore, E.A., Amanna, I.J., Rynko, A.E., Mori, M., Chen, Z., Slifka, M.K., 2016. Durability of vaccine-induced immunity against tetanus and diphtheria toxins: a cross-sectional analysis. Clin. Infect. Dis. 62, 1111–1118. Hunolstein, C., Alfarone, G., Scopetti, F., Pataracchia, M., Valle, R.L., Franchi, F., Pacciani, L., Manera, A., Giammanco, A., Farinelli, S., Engler, K., Zoysa, A.D., Efstratiou, A., 2003. Molecular Epidemiology and Characteristics of Corynebacterium diphtheriae and Corynebacterium ulcerans Strains Isolated in Italy During the 1990s. 52. pp. 181–188. Kaji, A., Parker, D.M., Chu, C.S., Thayatkawin, W., Suelaor, J., Charatrueangrongkun, R., Salathibuppha, K., Nosten, F.H., McGready, R., 2016. Immunization coverage in migrant school children along the Thailand-Myanmar border. J. Immigr. Minor.
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Contents lists available at ScienceDirect
Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
Research paper
Molecular epidemiology and antimicrobial susceptibility of outbreakassociated Corynebacterium diphtheriae in Thailand, 2012
T
Wantana Paveenkittiporna, Saowalak Sripakdeea, Onchuda Koobkratoka, Somchai Sangkitporna, ⁎ Anusak Kerdsinb, a b
National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Nonthaburi, Thailand Faculty of Public Health, Kasetsart University Chalermphrakiat Sakon Nakhon Province Campus, Sakon Nakhon, Thailand
A R T I C LE I N FO
A B S T R A C T
Keywords: Antimicrobial susceptibility Corynebacterium diphtheriae MLST Outbreak Thailand
Infections caused by Corynebacterium diphtheriae remain endemic in many countries. Since the implementation of the DTP (Diphtheria-Tetanus-Pertussis) vaccination program in 1977, only sporadic diphtheria cases have been reported in Thailand. In 2012, a diphtheria outbreak occurred in rural Thailand and 38 cases were reported, with the majority being adults (mean 22.1 years, range 5–72 years). The current study determined the genetic diversity of C. diphtheriae isolated from 83 individuals associated with either sporadic (n = 34) from 1994, 1996, 1997, 1998, 1999, 2000, 2012, and 2018, or 2012 outbreak (n = 49) diphtheria occurrences in Thailand. Antimicrobial susceptibility testing was performed on 41/83 isolates using broth microdilution. All sporadic (n = 27) and epidemic (n = 14) C. diphtheriae isolates (41/41; 100%) were susceptible to erythromycin (≤0.5 μg/ml), clindamycin (≤0.5 μg/ml), gentamicin (≤ 4 μg/ml), ciprofloxacin (≤1 μg/ml), and vancomycin (2 μg/ml), except tetracycline with a resistance rate of 34.1% (14/41 isolates). All isolates were intermediately resistant to penicillin (MIC range, 0.25–2 μg/ml). Multilocus sequence typing (MLST) revealed 17 sequence types (STs) among 83C. diphtheriae isolates. For the 2012 outbreak isolates, the predominant ST was ST243 (n = 34/ 49; 69.4%), followed by ST245 (n = 5/49; 10.2%) and ST244 (n = 4/49; 8.1%), whereas the main STs among the sporadic isolates were ST248 (n = 15/34; 44.1%), followed by ST209 (n = 7/34; 20.6%) and ST258 (n = 3/ 34; 8.8%). The ST243 outbreak strain was a single-locus variant of sporadic ST258. Phylogenetic analysis using concatenated sequences of 7 MLST genes from 17 STs revealed that ST243, ST248, and ST258 were located in the same cluster and ST243 appeared to have evolved from ST258, an endemic strain. This study highlights the importance of epidemiological surveillance together with characterization of C. diphtheriae strains to help inform the future control and prevention of diphtheria.
1. Introduction Diphtheria is a longstanding disease caused by toxigenic strains of Gram-positive Corynebacterium diphtheriae and the introduction of the diphtheria toxoid vaccine in 1923 and widespread mass immunization in the 1940s and 1950s led to the near elimination of the disease in the industrialized world (Efstratiou and George, 1996). Nonetheless, diphtheria remains endemic and has re-emerged in many developing countries around the globe, such as in Eastern Europe, Southeast Asia, South America, and the Indian subcontinent (Mattos-Guaraldi et al., 2003). Thailand's National Expanded Programme on Immunization (EPI), focusing on diphtheria, pertussis, tetanus, poliomyelitis, measles and tuberculosis, was initiated in 1977. By 2018, diphtheria, tetanus,
⁎
pertussis (DTP) vaccine coverage in Thailand was estimated to be 97% among infants below one year of age (World Health Organization, 2019). The DTP vaccine coverage in migrant school children on the Thailand-Myanmar border had the poorest coverage among children < 7 years of age with 89.2, 76.2, and 63.8% for the first, second, and third doses, respectively (Kaji et al., 2016). Booster of tetanus and diphtheria containing vaccine (Td) among age group ≥7 years showing 97.8%, 91.9%, 60.7% coverage for Td1, Td2 and Td3 vaccination, respectively (Kaji et al., 2016). According to a World Health Organization report, the numbers of cases for Thailand in 1996, 1997, 1998, 1999, and 2000 were 53, 38, 43, 52, and 15, respectively (World Health Organization, 2018). In June 2012 a diphtheria outbreak occurred in Loei province, northeast Thailand resulting in 38 cases and 2 cases were died. The first
Corresponding author at: Faculty of Public Health, Kasetsart University Chalermphrakiat Sakon Nakhon Province Campus, Sakon Nakhon 47000, Thailand. E-mail address: [email protected] (A. Kerdsin).
https://doi.org/10.1016/j.meegid.2019.104007 Received 4 May 2019; Received in revised form 12 August 2019; Accepted 15 August 2019 1567-1348/ © 2019 Elsevier B.V. All rights reserved.
Infection, Genetics and Evolution 75 (2019) 104007
W. Paveenkittiporn, et al.
tetracycline were determined among 41 out of the 83 isolates (sporadic = 27; outbreak = 14) using broth microdilution and results were interpreted according to Clinical Laboratory Standards Institute guidelines (CLSI, 2015). These MIC tested isolates were selected as representative STs that cover 17 STs distribution in the study.
case was a 40-year-old immunocompromised alcoholic male, who subsequently died of acute myocarditis. The second case was a 25-yearold male from a nearby village; subsequent cases were reported in a village close to where the second case lived and then spread to neighboring provinces. The Ministry of Public Health stated that during June 2012–January 2013, 38 laboratory-confirmed cases, 5 probable cases, and 93 asymptomatic carriers were reported from northeastern Thailand and the majority of confirmed cases in the 2012 outbreak were reported among adults (Wanlapakorn et al., 2014). This study determined the genetic relationship and antimicrobial susceptibility profiles among C. diphtheriae isolates from the 2012 outbreak, and from sporadic cases during 1994–2018 in Thailand.
2.3. Molecular characterization A multiplex PCR assay was performed for confirmation of C. diphtheriae based on dtxR (a species-specific marker), and for detection of strains carrying toxA and toxB (Pimenta et al., 2008). Multilocus sequence typing (MLST) was performed as described elsewhere (Bolt et al., 2010). MLST alleles and the resulting STs were assigned using the C. diphtheriae MLST database (https://pubmlst.org/cdiphtheriae/). eBURST was used to identify clonal complexes (CC) for these C. diphtheriae isolates and to compare ST relatedness to STs of other isolates currently available in the MLST database (Feil et al., 2004). Construction of phylogenetic trees for all STs in this study was performed via Phylogeny.fr (Dereeper et al., 2008).
2. Materials and methods 2.1. Isolates A total of 83 C. diphtheriae clinical isolates were characterized, with 49 (designated the outbreak group) isolated during the outbreak period in 2012 and 34 (designated the sporadic group) from sporadic cases that occurred throughout the country, from 1994 (n = 1), 1996 (n = 1), 1997 (n = 2), 1998 (n = 1), 1999 (n = 3), 2000 (n = 1), 2012 (n = 21; different region of outbreak), and 2018 (n = 4) (Table 1).
3. Results In total, identification of 83 C. diphtheriae strains was confirmed using conventional biochemical tests and PCR as described above. We characterized two groups of C. diphtheriae isolates as either sporadic or belonging to the 2012 outbreak. The sporadic group (n = 34) was composed of 30 toxigenic and 4 non-toxigenic isolates while the 2012 outbreak group (n = 49) from Loei and nearby provinces including Nong Bua Lamphu, Phetchabun, and Udon Thani consisted of 37 toxigenic and 12 non-toxigenic isolates based on Elek's test. Therefore, in total, there were 67 toxigenic and 16 non-toxigenic isolates and all were concordant with the PCR assay results for toxA and toxB as described above (Table 1). Based on the Clinical Laboratory Standards Institute guidelines (CLSI M45, 2015), antimicrobial susceptibility testing revealed that both sporadic and outbreak C. diphtheriae including toxigenic and nontoxigenic isolates (n = 41) were susceptible to erythromycin, clindamycin, vancomycin, gentamicin, and ciprofloxacin used in this study, except tetracycline which had a resistance rate of 34.1% (Table 2). The majority of outbreak isolates (11/14; 78.6%) were resistant to tetracycline whereas 11.1% of the sporadic isolates (3/27) were resistant to tetracycline (Table 2). Susceptibility testing of all isolates were intermediately resistant to penicillin (MIC range 0.25–0.5 μg/ml). MLST revealed 17 STs in the C. diphtheriae isolates. The predominant STs among the 2012 outbreak isolates were ST243 (n = 34; 69.4%), followed by ST245 (n = 5; 10.2%) and ST244 (n = 4; 8.1%) (Table 1, Fig. 1), whereas the predominant STs in the sporadic isolates were ST248 (n = 15; 44.1%), followed by ST209 (n = 7; 20.6%) and ST258 (n = 3; 8.8%) (Table 1, Fig. 1). All of the ST243 and ST209 isolates were toxin-producing whilst 86.6% (13/15) of sporadic ST248 isolates were toxin-producing (Table 1). The eBURST analysis showed that outbreak-associated ST243 (allelic profile: 2,4,8,19,7,3,9) is a single-locus variant of sporadic ST258 (allelic profile: 2,4,8,1,7,3,9). ST246 (allelic profile: 2,1,45,46,5,16,9) and ST209 (allelic profile: 3,3,4,2,3,3,3) are single-locus variants of ST254 (allelic profile: 2,1,71,46,5,16,9) and ST255 (allelic profile: 3,3,4,47,3,3,3), respectively (Figs. 1 and 2). The phylogenetic tree constructed using concatenated sequences of 7 MLST genes from the 17 STs revealed that ST248, ST258, and ST243 were located in the same cluster and that ST243 and ST258 appear to have evolved from ST248, an endemic strain found throughout Thailand (Fig. 2).
2.2. Microbiological methods Conventional biochemical tests were used to confirm C. diphtheriae identification (Thompson et al., 1983). Briefly, after culturing on sheep blood agar for 18–24 h at 37 °C, the organisms were tested using Gram stain, catalase production, reduction of nitrate, urea hydrolysis, starch hydrolysis, and production of acid in glucose, maltose, and sucrose (Thompson et al., 1983). Elek's test was conducted to detect the exotoxin produced by clinical isolates (Efstratiou and Maple, 1994). The minimum inhibitory concentrations (MIC) of penicillin, erythromycin, vancomycin, clindamycin, gentamicin, ciprofloxacin, and Table 1 Characteristics of C. diphtheriae from Thailand (n = 83). Category
Sequence type
Year of isolation
Toxin production
No. of isolates
Outbreak
123 209 243 244
2012 2012 2012 2012
245
2012
246 247 259
2012 2012 2012
Negative Positive Positive Positive Negative Positive Negative Negative Negative Negative
248
2012
2 1 34 1 3 1 4 1 1 1 49 9 2 4 1 1 1 4 2 1 1 1 1 1 1 1 1 1 1 34
Total Sporadic
209
255 258
243 249 253 254 256 257 285 Total
2018 1998 1999 2000 2012 2012 1994 1996 1997 1997 2012 2012 2012 1999 1999 2012
Positive Negative Positive Positive
Positive Positive
Positive Positive Negative Negative Positive Positive Positive
4. Discussion The genetic diversity and antimicrobial susceptibility among C. diphtheriae isolates associated with the 2012 outbreak and from 2
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Table 2 Antimicrobial susceptibility of C. diphtheriae (n = 41) determined by broth microdilution. Categories
Outbreak
Total Sporadic
Sequence types
Toxin production
243 244 245 123 209 246 247 259
Positive Positive Positive Negative Positive Negative Negative Negative
248
Positive Negative Positive Positive Positive Positive Positive Negative Negative Positive Positive Positive
209 255 258 243 249 253 254 256 257 285 Total
No. of isolates
7 1 1 1 1 1 1 1 14 8 2 5 2 3 1 1 1 1 1 1 1 27
Antimicrobial susceptibility testing Penicillin
Clindamycin
Vancomycin
Tetracycline
Erythromycin
Gentamicin
Ciprofloxacin
I I I I I I I I
S S S S S S S S
S S S S S S S S
R R R R S S R S
S S S S S S S S
S S S S S S S S
S S S S S S S S
I I I I I I I I I I I I
S S S S S S S S S S S S
S S S S S S S S S S S S
S S S S S S R R S S R S
S S S S S S S S S S S S
S S S S S S S S S S S S
S S S S S S S S S S S S
S = Susceptible; I = Intermediate; R = Resistant.
tetracycline has been reported in C. diphtheriae isolates from Brazil (12.8%) and Algeria (71.3%) (Pereira et al., 2008; Benamrouche et al., 2016). Susceptibility to penicillin, the first drug of choice, in all isolates in this study had not changed over time based on the intermediate level. Benamrouche et al. (2016) reported that 57.3% of 157 C. diphtheriae isolates from Algeria between 1992 and 2015 were intermediate for penicillin G. In Brazil, 14.8% of the 47 C. diphtheriae isolates were resistant to penicillin G (Pereira et al., 2008). The majority (7/11) C. diptheriae strains from Italy were intermediately resistant to penicillin (Hunolstein et al., 2003). Accordingly, our findings and previous
sporadic cases during 1994–2012 and 2018 were determined in this study. The outbreak was predominantly due to ST243 whereas the main STs among the sporadic isolates were ST248, however, both STs are closely related. Both toxigenic and non-toxigenic C. diphtheriae isolates in this study were susceptible to almost all antibiotics, except tetracycline and penicillin that showed reduced susceptibility. There were no differences in the antibiotic susceptibility patterns between the toxigenic and non-toxigenic isolates. Resistance to tetracycline was higher among the 2012 outbreak isolates compared to the sporadic isolates. Reduced susceptibility to
Fig. 1. eBURST analysis of sequence types (n = 623 STs) submitted to the C. diphtheriae MLST database (accessed on Apr 15, 2019). Outbreak and sporadic isolate sequence types from Thailand are shown as red and green dots, respectively. Clonal complexes and predicted founding STs are indicated by blue dots. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 3
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Fig. 2. Unrooted tree based on the alignments of concatenated sequences in 17 sequence types of C. diphtheriae from Thailand using the neighbour-joining method. Scale bar indicates sequence dissimilarity.
not able to compare the characteristics of the outbreak strains between Lao PDR and Thailand, although the outbreak occurred in the same period. Sornbundit et al. (2017) simulated a mathematical model to explain diphtheria transmission dynamics in Thailand (Sornbundit et al., 2017). Their simulations predicted outbreaks in the future could occur in approximately 2020 and 2045 (Sornbundit et al., 2017). The World Health Organization recommendation for diphtheria booster frequency is every 10 years after completing the primary dose (Scheifele and Ochnio, 2009). Although the rate of diphtheria toxin antibody loss was estimated to be 1/(15 × 365) day−1 (Hammarlund et al., 2016) and the overall seroprotection rate of diphtheria among Thai adults was 87.7–90.9% (Wanlapakorn et al., 2014), a booster vaccination every 10 years is recommended to maintain high protective vaccine-induced antibody titers for prevention of diphtheria in Thailand. This is essential as the number of susceptible hosts (especially in adults) is increasing which can lead to greater immunization waning. Moreover, Thailand's neighboring countries (Lao PDR, Cambodia, Myanmar) still have high incidences of diphtheria, which could spread across border. In addition, circulation of C. diphtheriae strains should be monitored for epidemiological control and prevention.
reports suggested that treatment with penicillin may not be effective; therefore, treatment with macrolides should rather be recommended in these regions. Among the STs in the outbreak and sporadic groups suggested that the 2012 outbreak C. diphtheriae isolates might be silently circulating in the area undetected (asymptomatic colonization) for a long time before an outbreak occurs. Several STs (ST123, ST243, ST244, ST245, ST246, ST247) were found in the same outbreak period. Among the outbreak strains, ST123 and ST245 were most closely related with 5 identical alleles (atpA, dnaE, fusA, odhA, rpoB) of the 7 MLST genes, whereas the remaining outbreak-associated STs were unrelated. ST243 (toxigenic isolates) were likely the first generation due to it being found throughout the epidemic in the outbreak area. This ST may possess better adaptive properties and abilities to disseminate in current populations. In addition, the majority of the cases were adults, so that waning vaccine-induced immunity may accelerate the infections (Wanlapakorn et al., 2014). However, it was not clear how ST243 emerged because almost all sporadic STs were different from the outbreak STs, with the exception of the ST243 (toxin-producing) strain which was an isolate from a sporadic event occurring 15 years ago (1997) in Chaiyabhum province (Table 1). This finding indicated that ST243 was present in Thailand before the outbreak in 2012. The MLST database revealed that ST243 has subsequently also been isolated in the UK in 2013 [https://pubmlst. org/cdiphtheriae/]. Phylogenetic analysis suggested that ST243 appeared to have evolved from ST258. Additionally, it should be noted that ST248 is an endemic strain in Thailand as this ST was identified throughout the country before and after the 2012 outbreak. However, ST248 has been found in Malaysia, the Philippines, and Germany (Khalid et al., 2019; https://pubmlst.org/cdiphtheriae/). During April 1, 2012–May 31, 2013, a diphtheria outbreak was reported in neighboring Lao PDR (Sein et al., 2016). However, of the 168 suspected cases reported from 7 of 17 provinces, 62 matched the clinical case definition and only 4 out of 21 cases were culture positive for toxigenic C. diphtheriae (Sein et al., 2016). Unfortunately, there was no report on microbiological or MLST characterization of the outbreak C. diphtheriae isolates in Lao PDR (Sein et al., 2016). Therefore, we were
Funding This study was supported by a grant from the Department of Medical Sciences, Ministry of Public Health.
Declaration of Competing Interest None.
Acknowledgement We thank the Kasetsart University Research and Development Institute (KURDI), Bangkok, Thailand for English editing assistance. 4
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