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For the direct detection of Group A Streptococcal Antigen



About Strep

 

Introduction

 

The genus Streptococcus was first identified by Louis Pasteur in 1879 as the bacteria responsible for puerpal sepsis1. Streptococcus pyogenes, also known as group A Streptococcus, causes disease of variable severity, contributing to 20% of tonsillopharyngeal infections2, and causing impetigo (pyoderma). Left untreated however, symptoms may become more severe with complications such as acute rheumatic fever, toxic shock-like syndrome and glomerulonephritis3. Rapid identification is therefore important to allow early treatment and prevent disease progression2.

 

Streptococcus pyogenes

 

Streptococcus pyogenes are gram positive, facultative anaerobic bacteria. They are often referred to as β-haemolytic bacteria which relates to the ‘halo’ that surrounds the individual colonies when grown on blood agar. This occurs as the bacteria produce the toxin streptolysin S, which gives the bacteria the ability to haemolyse red blood cells and damage cell membranes.  Membrane damage can also occur in lymphocytes, neutrophils, platelets, and cellular organelles such as lysosomes and mitochondria4. Streptococcus pyogenes also has a number of other virulence factors; the most important of which is the M protein. The M proteins resist phagocytosis by polynuclear leucocytes and therefore allow the organism to multiply rapidly in the host. Lipoteichoic acid is also expressed on the surface of Streptococcus pyogenes and is responsible for the binding of the organism to fibronectin which is present on the surface of oral epithelial-cell membranes. Mucoid strains of Streptococcus pyogenes are surrounded by a capsule that contains hyaluronic acid, which additionally contributes to the organisms ability to evade phagocytosis.3

 

As well as those virulent factors expressed on the cell surface, there are a number of extracellular substances Streptococcus pyogenes produce that also contribute to pathogenesis such as hyaluronidase, neuraminidase, DNases, streptokinase, pyrogenic exotoxins, streptolysin O and streptolysin S as described previously. Pyrogenic exotoxins for example are responsible for the rash in scarlet fever and render the patient more susceptible to endotoxic shock. Streptolysin O, DNases and hyaluronidase induce antibody formation approximately 10 to 17 days after infection5.

 

The majority of streptococci possess group specific antigens, which are usually carbohydrate structual components of the call wall. For Streptococcus pyogenes, the group specific antigen is a polymer of L-rhamose and N-acetyl-D-glucosamine. Clearview Exact Strep A detects this group specific carbohydrate antigen, thus confirming the presence of Group A streptococci (Strep A) .

 

(Reproduced by permission from Kenneth Todar):

 

http://textbookofbacteriology.net/streptococcus.html

 

Epidemiology

 

Strep A are a major cause of upper respiratory tract infections in humans. It is thought that 20% of all cases of tonsillopharyngitis are caused by group A streptococci2. It typically occurs in young children and infection rates are particularly high in environments such as schools, nursing homes and hospitals8,9. The incidence of infections caused by Strep A is believed to have re-emerged in the last 10-20 years5,9. In the past, in an effort to reduce complications such as rheumatic fever and glomerulonephritis, antibiotics have been prescribed in patients presenting with sore throats, even in those where there is no proven evidence that the cause is group A streptococci and the cause maybe viral2,10. This approach runs the risk of streptococci developing increased resistance to antibiotics. To ensure this does not happen, the use of rapid diagnostic tests is important.

 

Diagnosis

 

A specimen should be obtained by standard throat swab collection methods. The recovery of Strep A is dependent on the quality of the specimen collected. A tongue depressor and light should be used along with a good technique that swabs all areas at the back of the throat. This can increase the recovery of organisms ten-fold.

 

Culture:

 

Traditional methods for the identification of Strep A depend on the isolation and subsequent identification of the organisms. Throat swabs are cultured onto solid agar (e.g. tryptose or columbia agar) supplemented with either 5% horse or sheep blood11. The agar plates are then incubated for 18-24 hours at 37°C aerobically with the addition of 5% CO2 or anaerobically. Agar supplemeted with sheep blood has the advantage that it does not support the growth of Haemophillius haemolyticus which show similar colonial morphology when grown on sheep blood agar under aerobic conditions, therefore anaerobic conditions are favoured. Strep A are typically 0.5mm in diameter and are surrounded by a zone of complete haemolysis13.

 

The haemolytic action of streptococci on erythrocytes was first described by Brown in 1919. There are 4 recognised haemolysis patterns:

 

  1. Alpha haemolysis: Partial heamolysis observed around the colonies, the growth medium may be slightly discoloured.
  2. Beta haemolysis: Complete haemolysis observed with a clear, colourless zone surrounding colonies.
  3. No haemolysis: No apparent haemolysis or discoloration of the agar surrounding the colonies.
  4. Alpha-prime or wide zone alpha haemolysis: A small area of partially lysed cells next to the bacterial colony with a zone of complete haemolysis extending out into the medium.

 

Browns method has been used to characterise streptococcal groups from culture plates but it is limited in that other groups of streptococci also produce β-haemolytic colonies13.

 

Selective media can also be used for the isolation of Strep A e.g. SXT blood agar (blood agar containing sulfamethoxazole and trimethoprim). This media has limitations in that despite being a selective agar for Strep A, other streptococci may also grow in small numbers. Also a few strains of Strep A are susceptible to SXT and generally a second day of incubation is required for optimal recovery of Strep A14.

 

Following culture, all β-haemolytic organisms are confirmed with a streptococcal grouping kit.

 

Serological Testing:

 

Streptococci can also be identified according to their cell wall antigen, which is specific for each Streptococcus group. This classification system was first described by Rebecca Lancefield in 193314. There are many commercially available streptococcal kits including latex agglutination tests. The streptococcal group antigens are extracted from the cells (β-haemolytic colonies of streptococci) and their presence demonstrated with latex particles previously coated with group-specific antibodies. The latex particles will agglutinate in the presence of the homologous antigen, but no agglutination will occur in the presence of such antigen. The streptococcal grouping kits allow the identification of streptococcal groups, A, B, C, D, F and G.

 

Serological identification of Group A ß -Haemolytic Streptococcus by the M protein is the most specific marker and therefore regarded as the ‘gold standard’ for reliable strain identification11.

 

Rapid Immunoassay – Clearview Exact Strep A

 

Clearview Exact Strep A uses immunoassay technology to rapidly identify Group A Streptococcus direct from throat swab specimens. This method employs an extraction procedure, followed by a rapid test procedure, generating a visually read result.

 

The group A Streptococcal specific antigen is extracted using nitrous acid. The swab specimen is added to the extraction mixture (containing Reagents 1 and 2). Throat swab specimens should be collected using polyester tipped swabs.  No transport media is required as Clearview Exact Strep A is a rapid antigen test and therefore is not dependent on viable organisms. If a transport media is employed, use Liquid’s Stuart’s or Liquid Amies Transport Media. Do not use semi-solid Transport Media or Media containing charcoal.

 

If group A Streptococcus is present in the specimen, the nitrous acid (formed by the addition of Reagents 1 and 2) will break down the cell wall releasing the specific Strep A antigen (L-rhamose-N-acetylglucosamine).

 

Clearview Exact Strep A test is offered in two formats (cassette and dipstick). For the cassette format the extraction mixture is added to the Sample Well.  In the case of the dipstick format, the test strip is placed in the extraction mixture. The extraction mixture is allowed to react with the colloidal gold particles that have been coated with anti-group A Streptococcal antibodies.

 

If group A Streptococcus is present, antigen released from the cell wall during the extraction process binds to the antibody labelled colloid gold conjugate. This complex moves along the test strip by capillary action and binds to a region of immobilised rabbit anti-group A Streptococcus antibody in the Test Region, forming a pink line. If no antigen is present, the Test Region will remain clear.

 

Clearview Exact Strep A also provides an integral control feature. The appearance of a pink line in the Control Window shows the test has worked correctly.

 

References

 

  1. Efstratiou A. (2000) Group A streptococci in the 1990s. Journal of Antimicrobial Chemotherapy. 45,Topic T1, 3-12.
  2. Adam D. (2000) Group A beta-haemolytic streptococcal (GABHS) tonsillopharyngitis is still a common problem. Journal of Antimicrobial Chemotherapy. 45, Topic T1, 1-2.
  3. Bisno A. L. (1991) Group A Streptococcal Infections and Acute Rheumatic Fever. The New England Journal of Medicine. 325(11), 783-793.
  4. Nizet V., Beall B., Bast D.J., Datta V., Kilburn L., Low D.E. & De Azevedo J.C.S. (2000) Genetic Locus for Streptolysin S Production by Group A Streptococcus. Infection and Immunity. 68(7), 4245-4254.
  5. Kiselica D. (1994) Group A Beta-Haemolytic Streptococcal Pharyngitis: Current Clinical Concepts. American Family Physician. 49(5), 1147-1154.
  6. Heath A., DiRita V.J., Barg N.L. & Engleberg N.C. (1999) A Two-Component Regulatory System, CsrR-CsrS, Represses Expression of three Streptococcus Pyogenes Virulence factors, Hyaluronic acid capsule, Streptolysin S, and Pyogenic Exotoxin B. Infection and Immunity. 67(10), 5298-5305.
  7. Woods W.A., Carter C.T. & Schlager T.A. (1999) Detection of group A streptococci in children under 3 years of age with pharyngitis: Paediatric Emergency care. 15(5), 338-340.
  8. Schwartz B., Elliot J.A., Butler J.C., Simon P.A., Jameson B.L., Welch G.E. & Facklam R.R. (1992) Clusters of Invasive Group A Streptococcal Infections in family, hospital and nurse home settings: Clinical infectious Diseases. 15, 277-84.
  9. O’Brien K.L., Beall B., Barrett N.L., Cieslak P.R., Reingold A., Farley AM.M., Danila R., Zell E.R., Facklam R., Scwartz B. & Schuchat A. (2002) Epidemiology of Invasive Group A Streptococcus Disease in the United States, 1995-1999. Clinical Infectious Diseases. 35, 268-276.
  10. Little P.S., Williamson I. (1994) Are antibiotics appropriate for sore throats? Costs outweigh the benefits. British Medical Journal. 309, 1010-1011.
  11. Kaufhold A. & Ferrieri P. (1993) The Microbiological Aspects, Including Diagnosis, of β-Hemolytic Streptococcal and Enterococcal Infections. Laboratory Diagnosis of Infectious Diseases. 7(2), 235-256.
  12. Ross P.W. (1971) Throat Swabs and Swabbing Technique. The Practitioner. 207, 791-796.
  13. Facklam R.R. & Washington J.A. II (1991) Streptococcus and related catalase negative gram positive cocci: In: Balows A., Hausler W.J. Jr, Herrmann K.L., et al (eds): Manual of Clinical Microbiology, ed 5. Washington, DC, American Society for Microbiology: 238-257.
  14. Lancefield R.C. (1933) A Seriological Differentiation of Human and Other Groups of Hemolytic Streptococci. J.Exp.Med. 57, 571-593.
 


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