Biology Education

Supervisors: Lisa Wasserstrom, Microbiologist, PhD, at Clinical Microbiology, Region Skåne and David Nygren, Infectious Disease Physician, PhD, VO Infectious Diseases, SUS

Background

In descending order, the three most important bacteria that cause tonsillitis are S. pyogenes, F. necrophorum and S. dysgalactiae subsp. equisimilis (SDSE). Recently, we showed that S. pyogenes and F. necrophorum are equally associated with the development of complications in findings in tonsillitis (1), and F. necrophorum has been described as the most common cause of throat abscess (2), as well as an increasing cause of invasive infections in Sweden (3). S. pyogenes causes, in addition to complicated and invasive infections and the non-purulent complications, rheumatic fever and heart disease, but in high-income countries this rheumatic complication has almost disappeared as socioeconomics have improved (4-8). Rheumatic fever and heart disease continue to be a common cause of premature death and heart failure in low-income countries (4, 5, 7), with up to 4% of children found to have valvular heart disease secondary to throat infections with S. pyogenes at screening in low-income countries (7, 9 ). However, data on the prevalence of pathogens, including S. pyogenes, in throat infections in low-income countries are often lacking, and the science is flawed (6, 8). No study has investigated the presence of F. necrophorum as a cause of throat infections in any low-income country, but in studies from Nigeria, the bacterium has been seen as a potential cause of the orogangrenous and life-threatening infection noma, which mainly affects children suffering from starvation (10, 11).

Aim of the study

The aim of this study is to develop a multiplex PCR for F. necrophorum, S. pyogenes and SDSE. This PCR is planned to be used in research, and possibly as a clinical analysis at Clinical Microbiology. The primary purpose of the development is to cost-effectively analyze a longitudinally collected cohort of approx. 450 individuals and 5000 samples in The Gambia as well as for analyzes of a smaller study of 176 individuals with throat infection that were not sampled longitudinally but collected by the same study group (12).

Method and workplan

Singleplex PCR analyzes have been developed for these bacteria and are published (13-15) and at Clinical Microbiology real-time PCR is already used today for the detection of F. necrophorum. The project will begin by evaluating different primers and probes on known bacterial strains with the goal of creating a multiplex PCR where all three bacteria are included in the same real-time PCR. Next, the PCR method will be tested on 100 clinical samples divided in equal parts negative or positive for F. necrophorum, S. pyogenes or SDSE from PCR or culture identified in clinical routine and 20 samples with co-infection between F. necrophorum and beta-hemolytic streptococci . DNA from 200 µl of each sample is extracted and analyzed in singleplex (each PCR separately) and in multiplex (all three PCR reactions together). Results (1/0) and Cycle threshold (Ct) values ​​are compared between singleplex and multiplex PCR and cross-reactivity is evaluated among samples with co-infections.

The development of PCR is carried out as a 30 hp project. Laboratory supervision is carried out by Lisa Wasserstrom, Microbiologist, PhD, at Clinical Microbiology, Region Skåne and David Nygren, Infectious Disease Physician, PhD, VO Infectious Diseases, SUS. The opportunity to stay in The Gambia for setting up the developed method is given here, in collaboration with the Medical Research Council, The Gambia and the London School of Hygiene & Tropical Medicine. The method intends to be published, as well as the data from The Gambia in two separate publications.

References: 

1. Nygren D, Wasserstrom L, Holm K, Torisson G. Associations Between Findings of Fusobacterium necrophorum or beta-Hemolytic Streptococci and Complications in Pharyngotonsillitis-A Registry-Based Study in Southern Sweden. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America. 2023;76(3):e1428-e35.
2. Ehlers Klug T, Rusan M, Fuursted K, Ovesen T. Fusobacterium necrophorum: most prevalent pathogen in peritonsillar abscess in Denmark. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America. 2009;49(10):1467-72.
3. Nygren D, Holm K. Invasive infections with Fusobacterium necrophorum including Lemierre’s syndrome: an 8-year Swedish nationwide retrospective study. Clin Microbiol Infect. 2020;26(8):1089 e7- e12.
4. Carapetis JR, Mcdonald M, Wilson NJ. Acute rheumatic fever. The Lancet. 2005;366(9480):155-68.
5. Carapetis JR, Steer AC, Mulholland EK, Weber M. The global burden of group A streptococcal diseases. Lancet Infect Dis. 2005;5(11):685-94.
6. Gunnarsson R, Orda U, Elliott B, Heal C, Del Mar C. What is the optimal strategy for managing primary care patients with an uncomplicated acute sore throat? Comparing the consequences of nine different strategies using a compilation of previous studies. BMJ Open. 2022;12(4):e059069.
7. Ou Z, Yu D, Liang Y, Wu J, He H, Li Y, et al. Global burden of rheumatic heart disease: trends from 1990 to 2019. Arthritis Res Ther. 2022;24(1):138.
8. Gunnarsson RK, Ebell M, Centor R, Little P, Verheij T, Lindbaek M, et al. Best management of patients with an acute sore throat – a critical analysis of current evidence and a consensus of experts from different countries and traditions. Infect Dis (Lond). 2023;55(6):384-95.
9. Rothenbühler M, O’Sullivan CJ, Stortecky S, Stefanini GG, Spitzer E, Estill J, et al. Active surveillance for rheumatic heart disease in endemic regions: a systematic review and meta-analysis of prevalence among children and adolescents. The Lancet Global Health. 2014;2(12):e717-e26.
10. Falkler WA, Jr., Enwonwu CO, Idigbe EO. Isolation of Fusobacterium necrophorum from cancrum oris (noma). Am J Trop Med Hyg. 1999;60(1):150-6.
11. Enwonwu CO, Falkler WA, Phillips RS. Noma (cancrum oris). The Lancet. 2006;368(9530):147-56.
12. Armitage E, Keeley A, de Crombrugghe G, Senghore E, Camara F, Jammeh M, et al. Streptococcus pyogenes carriage acquisition, persistence and transmission dynamics within households in The Gambia (SpyCATS): protocol for a longitudinal household cohort study. Wellcome Open Res. 2023;8(41).
13. Nygren D, Brorson E, Musonda M, Wasserstrom L, Johansson A, Holm K. Geographical differences in tonsillar carriage rates of Fusobacterium necrophorum – A cross-sectional study in Sweden and Zambia. Anaerobe. 2021;69:102360.
14. Agerhäll M, Henrikson M, Johansson Söderberg J, Sellin M, Tano K, Gylfe Å, et al. High prevalence of pharyngeal bacterial pathogens among healthy adolescents and young adults. APMIS. 2021;129(12):711-6.
15. Jensen A, Hagelskjaer Kristensen L, Prag J. Detection of Fusobacterium necrophorum subsp. funduliforme in tonsillitis in young adults by real-time PCR. Clin Microbiol Infect. 2007;13(7):695-701.