Loop-mediated isothermal amplification for rapid detection of Streptococcus suis in hemoculture and clinical isolates

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Sorasak Intorasoot
Amornrat Intorasoot
Supaporn Limpaisarn
Nutsuda Kojorla
Santhasiri Orrapin
Manlika Wanmakok
Banyong Khantawa

Abstract

Introduction: Streptococcus suis is a zoonotic gram-positive cocci causing systemic infection and severe acute meningitis in humans. Infection is involved in the exposure of contaminated pigs and raw pork. Endemic usually occurs in northern area of Thailand where people eat raw pork-derived products. Due to lacking of effective vaccines, an early diagnosis of S. suis infection is extremely important for disease control and prevention.


Objective: To develop a rapid and high sensitive loop-mediated isothermal amplification (LAMP) technique for investigation of S. suis.


Materials and methods: Based on conserved sequences, glutamate dehydrogenase gene of S. suis was used as a target for LAMP primers design. The detection limit was compared with a conventional PCR. Thirty-four serotypes consisting of serotype 1-31, 32, 34 and 1/2 of S. suis were examined with the developed method. Specificity determination was tested with several blood-borne bacteria including other alpha and beta hemolytic Streptococcus spp. In addition, 25 clinical isolates and 30 positive hemocultures were resolved by established LAMP assay.


Results: LAMP assay exhibited approximately 1,000 times more sensitive than the conventional PCR with a final bacterial load of approximately 12 colony forming unit (CFU). Thirty-four serotypes of S. suis were tested and 24 of those – serotype 2-4, 6-12, 14, 15, 17-19, 21, 24-26, 28-31 and 1/2 could be detected by LAMP technique. The assay demonstrated a high specificity without cross-reactivity to other bacteria. In addition, 25 clinical isolates and 2 out of 30 cases from positive hemocultures were successfully amplified by this method.


Conclusion: LAMP assay developed is rapid and sensitive, and can be used for routine diagnosis of various serotypes of S. suis infection in clinical specimens.


Bull Chiang Mai Assoc Med Sci 2016; 49(2): 207-217. Doi: 10.14456/jams.2016.23

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How to Cite
Intorasoot, S., Intorasoot, A., Limpaisarn, S., Kojorla, N., Orrapin, S., Wanmakok, M., & Khantawa, B. (2016). Loop-mediated isothermal amplification for rapid detection of Streptococcus suis in hemoculture and clinical isolates. Journal of Associated Medical Sciences, 49(2), 207. Retrieved from https://he01.tci-thaijo.org/index.php/bulletinAMS/article/view/59892
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Research Articles

References

1. Kay R. The site of the lesion causing hearing loss in bacterial meningitis: a study of experimental streptococcal meningitis in guinea-pigs. Neuropathol Appl Neurobiol 1991; 17: 485-93.

2. Navacharoen N, Chabtharochavong V, Hanpasertpong C, Kangsanarak J, Lekagul S. Hearing and vestibular loss in Streptococcus suis infection from swine and traditional raw pork exposure in northern Thailand. J Laryngol Otol 2009; 123: 857-62.

3. Donsakul K, Dejthevaporn C, Witoonpanich R. Streptococcus suis infection: clinical features and diagnostic pitfalls. Southeast Asian J Trop Med Public Health 2003; 34: 154-8.

4. Mai NT, Hoa NT, Nga TV, Linh D, Chau TT, Sinh DX, et al. Streptococcus suis meningitis in adults in Vietnam. Clin Infect Dis 2008; 46: 659-67.

5. Okwumabua O, O' Connor M, Shull E. A polymerase chain reaction (PCR) assay for Streptococcus suis based on the gene encoding the glutamate dehydrogenase. FEMS Microbiol Lett 2003; 218: 79-84.

6. Staats JJ, Feder I, Okwumabua O, Chengappa MM. Streptococcus suis: past and present. Vet Res Commun 1997; 21: 381-407.

7. Hill JE, Gottschalk M, Brousseau R, Harel J, Hemmingsen SM, Goh SH. Biochemical analysis, cpn60 and 16S rDNA sequence data indicate that Streptococcus suis serotypes 32 and 34, isolated from pigs, are Streptococcus orisratti. Vet Microbiol 2005; 107: 63–9.

8. Kerdsin A, Dejsirilert S, Puangpatra P, Sripakdee S, Chumla K, Boonkerd N, et al. Genotypic profile of Streptococcus suis serotype 2 and clinical features of infection in humans, Thailand. Emerg Infect Dis 2011; 17: 835-42.

9. Nga TV, Nghia HD, Tu TP, Diep TS, Mai NT, Chau TT, et al. Real-time PCR for detection of Streptococcus suis serotype 2 in cerebrospinal fluid of human patients with meningitis. Diagn Microbiol Infect Dis 2011; 70: 461-7.

10. Wertheim HF, Nghia HD, Taylor W, Schultsz C. Streptococcus suis: an emerging human pathogen. Clin Infect Dis 2009; 48: 617-25.

11. Takamatsu D, Wongsawan K, Osaki M, Nishino H, Ishiji T, Tharavichitkul P, et al. Streptococcus suis in humans, Thailand. Emerg Infect Dis 2008; 14: 181-3.

12. Lun ZR, Wang QP, Chen XG, Li AX, Zhu XQ. Streptococcus suis: an emerging zoonotic pathogen. Lancet Infect Dis 2007; 7: 201-9.

13. Goyette-Desjardins G, Auger JP, Xu J, Segura M, Gottschalk M. Streptococcus suis, an important pig pathogen and emerging zoonotic agent-an update on the worldwide distribution based on serotyping and sequence typing. Emerg Microb infect 2014; 3: e45.

14. Wangkaew S, Chaiwarith R, Tharavichitkul P, Supparatpinyo K. Streptococcus suis infection: a series of 41 cases from Chiang Mai University Hospital. J Infect 2006; 52: 455–60.

15. Donsakul K, Dejthevaporn C, Witoonpanich R. Streptococcus suis infection: clinical features and diagnostic pitfalls. Southeast Asian J Trop Med Public Health 2003; 34: 154-8.

16. Fongcom A, Prusakorn S, Netsirawan P, Pongprasert R, Onsibud P. Streptococcus suis infection: a prospective study in northern Thailand. Southeast Asian J Trop Med Public Health 2009; 40: 511–7.

17. Wangsomboonsiri W, Luksananun T, Saksornchai S, Ketwong K, Sungkauparph S. Streptococcus suis infection and risk factors for mortality in northern Thailand. J Infect 2008; 57: 392-6.

18. Palmieri C, Varaldo PE, Facinelli B. Streptococcus suis, an emerging drug-resistant animal and human pathogen. Front Microbiol 2011; 2: 235.

19. Tarradas C, Arenas A, Maldonado A, Luque I, Miranda A, Perea A. Identification of Streptococcus suis isolated from swine: proposal for biochemical parameters. J Clin Microbiol 1994; 32: 578-80.

20. Ju Y, Hao HJ, Xiong GH, Geng HR, Zheng YL, Wang J, et al. Development of colloidal gold-based immunochromatographic assay for rapid detection of Streptococcus suis serotype 2. Vet Imm Immunopathol 2010; 13: 207-11.

21. Yang J, Jin M, Chen J, Yang Y, Zheng P, Zhang A, et al. Development and evaluation of an immunochromatographic strip for detection of Streptococcus suis type 2 antibody. J Vet Diagn Invest 2007; 19: 355-61.

22. Kerdsin A, Akeda Y, Hatrongjit R, Detchawna U, Sekizaki T, Hamada S, et al. Streptococcus suis serotyping by a new multiplex PCR. J Med Microbiol 2014; 63: 824-30.

23. Marois C, Bougeard S, Gottschalk M, Kobisch M. Multiplex PCR assay for detection of Streptococcus suis species and serotypes 2 and 1/2 in tonsils of live and dead pigs. J Clin Microbiol 2004; 42: 3169-75.

24. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al. Loop mediated isothermal amplification of DNA. Nucleic Acids Res 2000; 28: e63.

25. Huy NT, Hang le YT, Boamah D, Lan NT, Thanh P, Watanabe K, et al. Development of a single-tube loop-mediated isothermal amplification assay for detection of four pathogens of bacterial meningitis. FEMS
Microbiol Lett 2012; 337: 25-30.

26. Zhang J, Zhu J, Ren H, Zhu S, Zhao P, Zhang F, et al. Rapid visual detection of highly pathogenic Streptococcus suis serotype 2 isolates by use of loop-mediated isothermal amplification. J Clin Microbiol 2013; 51: 3250-6.

27. Arai S, Tohya M, Yamada R, Osawa R, Nomoto R, Kawamura Y, et al. Development of loop-mediated isothermal amplification to detect Streptococcus suis and its application to retail pork meat in Japan. Int J Food Microbiol 2015; 208: 35-42.

28. Okwumabua O, Persaud JS, Reddy PG. Cloning and characterization of the gene encoding the glutamate dehydrogenase of Streptococcus suis serotype 2. Clin Diagn Lab Immunol 2001; 8: 251-7.

29. Hill J, Beriwal S, Chandra I, Paul VK, Kapil A, Singh T, et al. Loop-mediated isothermal amplification assay for rapid detection of common strains of Escherichia coli. J Clin Microb 2008; 46: 2800-4.

30. Okada K, Chantaroj S, Taniguchi T, Suzuki Y, Roobthaisong A, Puiprom O, et al. A rapid, simple, and sensitive loop-mediated isothermal amplification method to detect toxigenic Vibrio cholerae in rectal swab samples. Diagn Microbiol Infect Dis 2010; 66: 135-9.

31. Brousseau R, Hill JE, Prefontaine G, Goh SH, Harel J, Hemmingsen SM. Streptococcus suis serotypes characterized by analysis of chaperonin 60 gene sequences. Appl Environ Microbiol 2001; 67: 4828-33.

32. Tien HT, Nishibori T, Nishitani Y, Nomoto R, Osawa R. Reappraisal of the taxonomy of Streptococcus suis serotypes 20, 22, 26, and 33 based on DNA–DNA homology and sodA and recN phylogenies. Vet Microbiol 2013; 162: 842-9.