You are on page 1of 5


Journal of Medical Microbiology, (2006) 24 (2):92-6

Review Article

*KN Brahmadathan, P Gladstone

Post-streptococcal sequelae, especially acute rheumatic fever/rheumatic heart disease continue to occur in significant
proportions in many parts of the world. Despite several attempts with various intervention strategies, little success has
been achieved in the control of acute rheumatic fever/rheumatic heart disease in India. The success of the control programmes
depends upon timely primary prophylaxis with benzathine penicillin for which a microbiological confirmation of group
A streptococcal pharyngitis is essential. Isolation of beta hemolytic streptococci from throat cultures and their identification
as GAS in the laboratory, clinches the microbiological diagnosis while demonstration of a ‘significant rise’ in antibody
titers such as Anti-streptolysin O and Anti-deoxyribonuclease B differentiates it from a group A streptococcal carrier
state or pharyngitis of a viral etiology. Despite the easiness with which these can be achieved, many laboratories in India
are not equipped to do so. Enhancing bacteriological and serological facilities in laboratories across the country will
drastically improve the clinician’s ability to diagnose bonafide GAS pharyngitis and help to institute penicillin prophylaxis
at the appropriate time. This will go a long way in enhancing the compliance to penicillin prophylaxis which is the
cornerstone of any RF/RHD control program.
Key words: Group A Streptococci (GAS), Pharyngitis, Post-streptococcal sequelae, RF/RHD, ASO, ADNB

Rheumatic fever (RF) and rheumatic heart disease (RHD)
continue to be a problem of major concern in most developing
countries including India.1-6 Given a prevalence rate of 4 to 6
per 1000 children per year,7 there is approximately 1 to 1.25
million cases of RF/RHD in India today. Despite sporadic
reports of a declining trend in the incidence of RF/RHD,8 these
two complications continue to occur in endemic proportions
and engage cardiologists and cardiac surgeons alike. Apart
from the increased morbidity and mortality due to these
conditions, the high cost of surgical intervention for the
treatment of later complications of RF/RHD puts heavy
economic burden on the families of affected children. Timely
diagnosis and treatment of streptococcal pharyngitis with
benzathine penicillin therefore acquires special significance in
the prevention of RF/RHD in endemic communities.
A microbiological diagnosis of clinically suspected
pharyngitis is essential for the confirmation of a bona fide
group A streptococcal (GAS) infection.9,10 About two-thirds
of all clinical pharyngitis is caused by viruses, often selflimiting and for which no effective treatment exists. On the
other hand, adequate treatment of streptococcal pharyngitis
with benzathine penicillin is the method of choice for the
prevention of post-streptococcal sequelae. Therefore,
differentiating streptococcal pharyngitis from that of a viral
*Corresponding author (email: <>)
Department of Clinical Microbiology, Christian Medical College,
Vellore - 632 004, Tamil Nadu, India
Received : 07-07-05
Accepted : 14-12-05

etiology will help the clinician to administer penicillin to the
affected child at the appropriate time.
Clinical Features
In general, the clinical features of GAS pharyngitis are not
specific and cannot be easily differentiated from that of nonstreptococcal pharyngitis; therefore, sole diagnosis on clinical
grounds is highly improbable. However, various clinical and
epidemiological factors may narrow down the diagnosis,
which may be further confirmed by laboratory methods.11,12
Epidemiologically, GAS pharyngitis is often seen in children
between 5 and 15 years of age and tends to occur in colder
months of the year. Patients with GAS pharyngitis often
complain of pain while swallowing, fever, enlarged cervical
lymph nodes and fatigue. Headache, nausea, vomiting and
abdominal pain may be seen especially in children. Tonsils are
reddened and swollen. In acute cases, the roof of the mouth
may have fine petechial lesions.13 Although none of these are
specific for GAS pharyngitis, absence of fever or presence of
clinical features such as cough, hoarseness and running nose
are common symptoms of viral upper respiratory infections.13
Clinical Specimens
The success in isolating GAS in culture or getting a
positive result with a rapid antigen detection test lies in
collection of a well-taken throat swab. Two swabs may be
rubbed well over both the tonsils and posterior pharyngeal
wall, taking care not to touch the oral cavity or any other
oropharyngeal region.9,13 Specimens should be collected prior
to any antibiotic treatment and processed in the laboratory

Isolation and identification of Group A β -hemolytic streptococci (GAS) Laboratory diagnosis of streptococcal pharyngitis depends upon the successful isolation of β-hemolytic streptococci (BHS) and its identification as GAS. Identification of GAS Bacitracin susceptibility In many laboratories bacitracin susceptibility test is the method of choice to identify GAS. Therefore. while producing them in routine diagnostic laboratories requires certain expertise and is labor intensive. The rapidity with which GAS is identified in a throat culture isolate is important because delay in diagnosis may mean delay in treatment and this could unnecessarily predispose the child to post-streptococcal sequelae. but in Indian situation. In general GAS can be identified on the same day if BHS are well isolated on the BA plate. Pre-sterilized quality BA plates are either not easily available in the Indian market or they are expensive. Many laboratories circumvent this problem by using outdated human blood obtained from blood bank. 100 µl of group A antiserum is added to 1 ml of 10% Staph.13 This enhances the patient compliance to treatment and has been shown to significantly increase the number of patients appropriately treated for GAS pharyngitis. Major advantage of this system is that highly potent antisera are diluted and therefore used in small quantities during preparation of the co -agglutination reagent. Rapid Antigen Detection Tests (RADTs) Introduced in the 1980s. More recently. This test has a sensitivity of >95%. co-agglutination.13 Therefore a negative www. This is neither scientifically acceptable nor satisfactory since human blood often contains antibiotics and other anti bacterial substances including antistreptococcal antibodies that inhibit the growth of GAS. Since we use only 50 µl of the reagent for identification of each strain. most of them have sensitivity between 70% and 90%.16 Batch to batch variation may occur in the commercial discs and therefore it is essential to test each batch for quality control with known GAS strains. RADTs have a basis of extracting carbohydrate antigen from BHS and identify them by immunological methods. 15.9 In itself this is not a difficult task. Unavailability of grouping antisera used for grouping of BHS is a deterrent for many laboratories to identify GAS. which therefore is recommended for the isolation and characterization of BHS/GAS. these antisera will remain potent for many years. Maintaining a sheep pen for the purpose of collecting sterile sheep blood for preparation of BA plates can be both expensive and exasperating. provided one has easy access to quality sheep blood agar plates (BA). as compared to commercial products. Grouping can be done on the organism isolated from throat cultures or extracts prepared directly from throat cultures.April 2006 Brahmadathan & Gladstone – Microbiological Diagnosis of GAS Pharyngitis without any delay. When performed together with the co 93 agglutination reagents.ijmm. Intravenous immunization of locally available rabbits with heat-killed formalin inactivated group A cells result in the production of high potency antisera. the homemade reagents work out to be extremely cheap and can be used to detect GAS from BA or broth cultures. they keep well for several weeks under ordinary refrigeration. this technique can identify a GAS strain from a BA plate in about 30-45 minutes. liposomal and optical immunoassays have been employed for this purpose. Commercially available antisera are exorbitantly costly. In our experience such homemade antisera are of high quality and highly . one can resort to GAS identification by this method. they are more difficult to obtain than sheep blood.19 and have used them for group identification since then. provided one takes extra precautions to ensure absolute sterility of blood during collection. We first produced such co agglutination reagents in our laboratory to test groups A. F and G in 197718. 17 of which the time tested Lancefield’s hot-acid extraction technique and Fuller’s formamide extraction method are the most widely used. due to antigenic cross-reactions. Numerous methods are available in the laboratory for this.9 Identification of GAS by grouping is a specific method although there are isolated cases where group A has been identified as group G or vice-versa. If this is done regularly. Collecting sheep blood from the local slaughterhouses is an eminently viable proposition. aureus Cowan 1 cells and then diluted to 10 mL with phosphate buffer. In our laboratory. For example. use of animals for antisera production has been restricted for ethical considerations.9 The micro-nitrous acid extraction procedure of El Kholy17 is a rapid method that can be standardized in any routine diagnostic laboratory with ease. but with a specificity of >95% with culture as a gold standard. these antisera can easily be modified into cost-effective co agglutination or latex particle based reagents. they can provide a result within a few hours even when done directly on throat cultures.14 Using horse or ox blood is also recommended. Otherwise it takes an additional 24 hours for the final identification. Blood sample may be collected for antibody tests. one can test approximately 200 strains with 10 ml reagent. a practice that has been followed till date. C. enzyme immunoassays. If properly preserved. grouping antisera were first prepared in early 1960’s. therefore most laboratories prepare them in their own laboratory.20 Although tests such as latex agglutination. If properly preserved at +4oC with added merthiolate. Group identification The recommended method of GAS identification is by testing β-hemolytic colonies on BA for group A specific carbohydrate antigen (ACHO). is only a presumptive test and is not recommended since group G and C streptococci can give false positive results. Despite their higher cost. B. Once produced.

recently we changed over to nephelometric titration. cumbersome and time consuming. titer obtained with a single serum sample can be interpreted based on a cut-off value defined as the upper limit of normal (ULN). Practical difficulties in getting two serum samples from children and the time taken to demonstrate a four-fold rise in titer make this unfeasible on a routine basis. Evaluation of such kits in countries where group C and G are also endemic has shown high degree of false positivity (personal observations). we were determining ASO and ADNB titers by the conventional micro-titer technique using SLO and DNase B enzyme produced and standardized in our laboratory. Often this can be achieved within 24 hours of processing throat culture which will help the clinician to institute penicillin prophylaxis at the earliest. many laboratories may find it difficult to establish this technique for antibody titrations. resistance to other antibiotics such as macrolides is on the increase which can be monitored only by regular testing of GAS strains to such . oral antibiotics www. 22-24 Demonstration of a significant or four-fold rise in titer on paired serum samples taken at an interval of 7 to 14 days apart will indicate an ongoing or an acute infection. Caution need be exercised while interpreting the titers for two reasons. GAS continues to be exquisitely susceptible to penicillin29. we have been monitoring antibiotic resistance in GAS since 197030 and this has helped us to detect a significant increase in erythromycin resistance since 1993. Interpretation of Cultures In countries where streptococcal infections are endemic. determination of antideoxyribonuclease B antibody (ADNB) is a valuable method of diagnosing GAS infection. For many years.9 On the other hand. there is no quality control on these kits after they are imported to India so that batch to batch variations do occur while using them in diagnostic laboratories. Therefore. 2 recommended in a clinical setting. ASO and ADNB are often used to: (1) confirm GAS infections where facilities for culture do not exist (2) to confirm doubtful positive rapid antigen detection test or (3) confirm a diagnosis of post-streptococcal etiology.22 Serological Diagnosis Historically. ULN represents the highest level of antibodies that can be observed in 20% of normal individuals who have demonstrable antibodies in them. Though molecular techniques have been standardized recently with improved sensitivity. determination of anti-streptolysin O antibodies (ASO) had been the mainstay of confirming a diagnosis of GAS pharyngitis as well as RF. More importantly. Use of commercially available kits to identify GAS directly from throat cultures has not become popular in India because of their cost.21 Therefore isolation of GAS from throat cultures should be interpreted with caution. Any ASO titer above these cut-off values will indicate a GAS infection. However. It is to be emphasized that ULN titers should be determined in different geographical areas because such titers are distinctly higher in endemic regions as compared to non-endemic areas. factors such as cost and feasibility has hindered their use in smaller laboratories. especially in a child with viral pharyngitis. are vol. such cases may result in over treatment with penicillin. In such instances. presence of GAS in throat in the absence of a significant rise in antibodies indicates a carrier state and no GAS infection.28. the equipment is expensive and therefore. which is an automated. This method is generally convenient and reliable although an antibody response from an earlier GAS infection may confuse the final interpretation of the current ASO titer. on a routine basis.30 which therefore is the drug of choice to treat the infections caused by them. Many laboratories in India now use commercial latex agglutination-based ASO kits for routine diagnostic purpose. One major disadvantage of ASO test is its inability to demonstrate an antibody response in many cases of impetigo. isolation of BHS and its identification as GAS in the presence of clinical symptoms of pharyngitis confirms its streptococcal etiology and needs no further confirmation by antibody tests.7 Though these tests performed well in our hands for many years.ijmm. Therefore. Since penicillin treatment depends upon a positive test. Secondly. Thus. Alternately. culture techniques. they are labor intensive. Antibiotic Susceptibility Testing Despite reported penicillin tolerance. 24. 25 In such cases. In our laboratory. which give direct evidence of GAS etiology and takes less time than antibody detection. 26 The ADNB test is more sensitive and the magnitude of antibody response is higher than that of ASO both in cases pharyngitis and impetigo as well as the non suppurative sequelae. simple and rapid method that does not require much technical expertise. Firstly. Firstly. Thus in practice it is not necessary to test their susceptibility on a routine basis. No. Laboratories using these kits should therefore test the quality of each batch of the kit using appropriate controls.94 Indian Journal of Medical Microbiology RADT should always be confirmed with a throat culture. for several reasons. However this cannot be recommended in highly endemic situations.31 Empirical treatment of clinical pharyngitis with various antibiotics including penicillins is a common practice in general practice. They should also determine their own cut-off titers for the kit by determining ULN for their population using the same brand of kit. however it is imperative to look for emergence of resistance to penicillin in our locality. determination of four-fold rise in titers of antistreptococcal antibodies can differentiate bona-fide GAS pharyngitis from GAS carrier state. pharyngeal carriage of GAS is a common event. the cut-off titers recommended in these kits are based on evaluation done in countries where GAS infections are less endemic than India.

Rizvi SF. Rheumatic fever and rheumatic heart disease. Given the magnitude of the problem of RF/RHD in India and the need for its prevention. Samad A.923:1-122. Indian Heart J 2003. 4.91:87­ 8. we have to upgrade our expertise to diagnose GAS infections in the laboratory. GAS should be identified by grouping techniques with either in-house or commercially prepared antisera. 2. Heart 2004. to patients who may later go on to develop RF/RHD due to lack of adequate antibiotic protection. Wannamaker LW. Differences between streptococcal infections of the throat and skin. Throat cultures should be collected with care as the success in getting reliable results relies on their proper collection.282:23-31. Prevalence of rheumatic and congenital heart disease in schoolchildren of Kathmandu valley in Nepal. ASO and ADNB may be used in patients for the confirmation of diagnosis. Raizada A. 5. N Engl J Med 1970. 34 We have recently standardized this method in our laboratory and result on typing of 227 GAS strains show high heterogeneity among GAS strains circulating in the community. Epidemiological Typing Typing of GAS strains based on T and M proteins are of epidemiological interest. Bahadur KC. 3. Pathogenesis of acute rheumatic fever and rheumatic heart disease: Evasive after half a century of clinical. it will be important for us to generate such data on the prevalence of M types in our community. 35. 6. Interpretation should be made in comparison with ULN as standardized for a particular population. Bull World Hlth Organ 1981.ijmm. Malla R. This is quite different from what one sees in non endemic temperate countries where a few M types are responsible for most of the invasive GAS infections. Geneva 1996. Indian Heart J 2003. Padmavati S. Gurung S. one may encounter larger number penicillin anaphylaxis which can significantly reduce patient’s compliance to antibiotic treatment. A simple score card for the tentative diagnosis of www. Breese BB. Kaplan EL.90:394-9. Conclusions In the final analysis. Aggarwal AK. World Health Organization. M typing technique has been revolutionized and almost 100% typing can be done with the modern gene sequencing method. 11. 9.54:54-8. 7.36 While most diagnostic laboratories will find it difficult to adopt this technology. Pasha O. Marsh DR. microbiological diagnosis of GAS pharyngitis can be a challenging task at every stage of its execution.9 With a GAS vaccine becoming a reality now than ever before. 10.55:615-8. Kumar R. et al. Koshi G. Rheumatic fever and rheumatic heart disease in India at the turn of the century. Shrestha MP.55:158-60. 2004. epidemiological and laboratory investigation. Emphasis on basic bacteriological techniques. Johnson DR. Antibiotic susceptibility testing though not mandatory may be very useful to detect erythromycin resistance and penicillin tolerance if any.April 2006 Brahmadathan & Gladstone – Microbiological Diagnosis of GAS Pharyngitis do not give as much coverage as benzathine penicillin. there is no doubt that bacteriology laboratories in India will have to play a much bigger role than they do now.33 With the advent of gene sequencing technology. Rheumatic fever and rheumatic heart disease in rural south Indian children.53:35-7. Indian Heart J 2004. data on the prevalence of M types in a community has acquired great significance. Bicova R. Until such times. Heart 2005. This will help us to assess whether a vaccine designed in the context of a nonendemic population will be equally effective in an endemic situation. If possible 95 epidemiological typing may be undertaken to determine the M types circulating in the community which may be useful in assessing or developing a vaccine at a later stage. Sramek J. it is better to institute the treatment after confirming a GAS etiology which would overcome the above . A communitybased rheumatic fever/rheumatic heart disease cohort: twelveyear experience. 8. For this. Khan MA. et al. Declining prevalence of rheumatic heart disease in rural schoolchildren in India: 2001-2002. 32 Conventionally. Bacitracin susceptibility could be used for routine identification of GAS if appropriate quality control is carried out with every batch of bacitracin discs. Cherian G. quality of the reagents and techniques as well as proper interpretation of results based on local conditions is the secret of its successful implementation. Dos and Doníts Physicians should identify the suspected cases of GAS pharyngitis with the given clinical and epidemiological features and send appropriate specimens for laboratory confirmation. Rajbhandari S. Kaplan EL. Ganguly NK. RADTs can be used for rapid diagnosis but all negative tests should be confirmed with throat culture. M typing was a tedious and almost impossible task and workers used to generate M type data extrapolated from the data obtained by T typing. Thus scientifically. Secondly. but should not be used to interpret when throat culture is negative. References 1. Sharma D. Indian Heart J 2001. Laboratory diagnosis of group A streptococcal infections. over treatment with penicillin may pave way to increased penicillin tolerance and subsequent penicillin resistance. Havlickova H. Havlicek J. Status of rheumatic heart disease in Pakistan. WHO Technical Report Series.59:599-603. Gomathi M. RF/RHD will continue to cause much morbidity and mortality because microbiological techniques form an integral part of their diagnosis. Finally. Kundi A. Sheep blood and not human blood should be used for preparing blood agar. Jose VJ. Benjamin V.

Ayoub EM. Brahmadathan KN. Thangavelu CP. Specimen collection. An outbreak of post-streptococcal reactive arthritis. Antibodies in acute rheumatic fever. Further studies on the reliability of the bacitracin inhibition test for the presumptive identification of Lancefield group A streptococci. 35. 16. 24. Wold AD. Liu M. Gwaltney JM.25:574­ 83.28:836-9. Fullerton KE. No. Texas. Adams GJ. J Clin Microbiol 1996. Am J Public Health Nat Hlth 1961. Gladstone P.79:479-81. Nelson J. Murray PR. 30. Kumar R.51:1872-92.188:818-27. Johnson DR. editors. Geneva. Clinical score card for diagnosis of group A streptococcal sore throat.7:847-54. Cunningham. Kaplan EL. Pediatr Infect Dis J 1999. In: Mackie & McCartney Practical Medical Microbiology. Koshi G. 33. Pandian R. Molecular genetic analysis of 675 group A Streptococcus isolates collected in a carrier study at Lackland Air Force Base. Indian J Pediatr 2002. J Infect Dis 2003. John TJ. Circulation 1960:21:598-614.11:335-7. Gackstetter GD. Increasing resistance among group A streptococci causing tonsillitis in a tertiary care hospital in southern India.71:709-12. Clin Microbiol Rev 2000. 311­ 12.59:1554-5. Koshi G. Koshi G. 764. Sequencing emm-specific PCR products for routine and accurate typing of group A streptococci.69:471-5. Bisno AL. Korula RJ. Krause RM. J Paediatr 1976. Anti-streptolysin O test by microtechnique Indian J Med Res 1971. Kaplan EL.89:576-9. Tebbutt GM. Hall MM.166:374-82. Anitha P. Wannamaker LW. Parker MT. Schwartz RH. Sridharan G. 1984. 19. Washington JA II. Microbiological confirmation of streptococcal pharyngitis. Peters JE. 18.147:205-8. Proc Soc Exp Biol Med 1974. Koshi G. Beta hemolytic streptococci in survey throat cultures in an Indian population. Reedbooks: Berkshire. Koshi G. Koshi G. 13. Ray P. Madhuri V. Changing pattern of antibiotic susceptibility of antigenic groups of streptococci. 17. Streptococcal antideoxyribonuclease B: Microtechnique determination. Fleisher GR. Diagnosis and management of group A streptococcal pharyngitis: A practice guideline.34:953-8. 1988. Nandi S. Indian J Med Res 1984. Beall B. 29. J Infect Dis 1992. Sivadasan K. El Kholy A. Kimura Y. Clinical evaluation of a latex agglutination test for streptococcal pharyngitis: performance and impact on treatment rates. Kotami S and Shiokawa Y Editors. 103. Indian J Med Res 1997.ijmm. The reliability and rapidity of the coagglutination technic and its comparison with precipitin technic in the grouping of streptococci. 25. Mathai E. www. Simmons A. Epidemiologic analysis of group A streptococcal serotypes associated with severe systemic infections. 32. Report of a WHO vol. 22. Brahmadathan KN.13:470-511. rheumatic fever or uncomplicated pharyngitis. 2 Study Group. International survey of distribution of serotypes of Streptococcus pyogenes. Collee JG. 24. 20. ICMR Bull 1982. World Health Organization. Bull WHO 1967. Grouping of beta hemolytic streptococci from primary plates by micronitrous acid-coagglutination method. Vanitha M. Amer J Clin Pathol 1979. Marmion BP. Kaplan EL. M. 23. 12. Hoe NP. 28. Thangavelu CP. Brahmadathan KN. Gerber MA.30:421-6. In: Recent Advances in Streptococci and Streptococcal Diseases. culture containers and media.96 Indian Journal of Medical Microbiology streptococcal pharyngitis. Horn DL. Pathogenesis of group A streptococcal infections. McGhie D.131:54-17s. Ayoub EM. Rheumatic fever and rheumatic heart disease. Del Roasario MC. 15. Ganguly NK. Lieu TA. J Lab Clin Med 1968.105:249-53. Clin Infect Dis 1997. Simplified extraction procedure for serological grouping of beta-hemolytic streptococci. p. Marr W. Appl Microbiol 1974. Stevens DL. 1996. Shwartz JS. 14. et al. Rajeswari . Coleman DJ. p. Clin Microbiol Infect 2005.37:513-27. Thompson T. WHO Technical report Series. Proceedings of the IXth Lancefield International Sympoisum on Streptococci and Streptococcal Diseases. 27. Facklam R. Pediatr Infect Dis J 1988. Collee JG. 34. 21. 36. Churchill Livingstone: Edinburgh. Streptolysin O. Susceptibility of group A beta-hemolytic streptococci to thirteen antibiotics: examination of 301 strains isolated in the United States between 1994 and 1997. San Antonio. Wannamaker LW. Myers RM. 26. No.18:1069-72. Am J Dis Child 1977. J Clin Pathol 1977. Wannamaker LW.71:867-73. Johsnon DR. Kaplan EL. Wannmaker LW.12:11-4. Vohra H. 31. Suppression of its antigencity by lipids extracted from skin. Fraser AG. Bacitracin differentiation of presumptive identification of group A âhemolytic stereptocci: Comparison of primary and purified plate testing. Brahmadathan KN.