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Education & Practice Online First, published on January 30, 2014 as 10.1136/archdischild-2013-304884 INTERPRETATIONS
How to use antistreptolysin O titre E S Sen, A V Ramanan
▸ The literature search strategy for this article and multiple choice questions are included in the online supplementary appendix. To view these files please visit the journal online (http://dx.doi.org/10.1136/ archdischild-2013-304884). Department of Paediatric Rheumatology, Bristol Royal Hospital for Children, Bristol, UK Correspondence to Dr ES Sen, Department of Paediatric Rheumatology, Bristol Royal Hospital for Children, Upper Maudlin Street, Bristol, BS2 8BJ, UK; [email protected] Received 15 September 2013 Revised 14 December 2013 Accepted 3 January 2014
To cite: Sen ES, Ramanan AV. Arch Dis Child Educ Pract Ed Published Online First: [ please include Day Month Year] doi:10.1136/archdischild2013-304884
ABSTRACT Group A streptococcus (GAS) is the cause of a wide range of acute suppurative and, following a latent period, non-suppurative diseases such as rheumatic fever and poststreptococcal glomerulonephritis. Diagnosis of the latter group requires evidence of preceding GAS infection. The bacteria produce a range of extracellular antigens, including streptolysin O, which induce an antibody response in the host. A rise in antistreptolysin O titre (ASOT) is indicative of preceding GAS infection. In clinical practice, often only a single ASOT measurement is available and its timing in relation to a possible GAS infection is unknown. Interpretation of the result in this context is liable to misdiagnosis. In order to optimise diagnosis of preceding GAS infection, at least two sequential ASOT measurements, together with simultaneous assay for anti-DNase B, a second antistreptococcal antibody, is recommended.
INTRODUCTION Group A Streptococcus (GAS; Streptococcus pyogenes) is a Gram-positive, β-haemolytic bacterium that can cause both acute suppurative disease and also non-suppurative complications in humans. The diseases caused by GAS are shown in table 1. They most commonly affect infants, children and young adults. The primary site of infection is frequently the skin or throat; indeed, streptococcal pharyngotonsillitis remains the commonest form of bacterial throat infection.3 Acute GAS infection may also present as scarlet fever, characterised by an erythematous rash starting on the head and neck before spreading to the trunk and limbs with onset 1–2 days after throat infection. There are 4–5 million cases of GAS infections each year in the USA alone, with higher incidence in developing countries.4 Non-suppurative complications of GAS infections can have significant morbidity and mortality. They include acute rheumatic fever (ARF), rheumatic heart disease (RHD), poststreptococcal
glomerulonephritis (PSGN), poststreptococcal reactive arthritis (PSRA) and paediatric autoimmune neuropsychiatric disorder associated with streptococcal infection (PANDAS). The pathogenesis of these conditions is thought to be shared epitopes on antigens from GAS and the host resulting in an autoimmune response.2 Onset of these complications is often several weeks after an acute infection when symptoms and signs may have resolved. In a significant number of cases, the initial infection is subclinical.5 Throat cultures taken at the time of presentation with rheumatic fever or nephritis may be negative because of clearance of GAS by the host immune system during the latent period5 or due to previous antibiotic treatment.6 Interpretation of throat cultures at the time of presentation with a possible poststreptococcal complication is further confounded because a swab positive for GAS may represent asymptomatic carriage and not infection. A meta-analysis of 18 studies reported the prevalence of GAS carriage among asymptomatic children to range from 3% to 26%, with a pooled prevalence of 12% (95% CI 9% to 14%).7 However, diagnosis of non-suppurative complications requires evidence of recent preceding streptococcal infection. This can be provided by serological evaluations, one of which is measurement of antistreptolysin O titre (ASOT). Other serological tests include antideoxyribonuclease B (anti-DNase B), antihyaluronidase and antistreptokinase antibodies.8 This paper aims to identify which children should have ASOT measured and address questions regarding the timing and interpretation of the results.
PHYSIOLOGICAL BACKGROUND (WHAT IS ANTISTREPTOLYSIN O?) When cultured on blood agar, GAS produces a clear area of haemolysis around its colonies. This results from secretion of proteins called streptococcal haemolysins,
Sen ES, et al. Arch Dis Child Educ Pract Ed 2014;0:1–8. doi:10.1136/archdischild-2013-304884 1 Copyright Article author (or their employer) 2014. Produced by BMJ Publishing Group Ltd under licence.
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Interpretations Table 1 Suppurative and non-suppurative presentations of GAS disease1 2 Suppurative
Non-suppurative
Tonsillopharyngitis
Acute rheumatic fever
Peritonsillar abscess
Rheumatic heart disease
Cervical lymphadenitis
Poststreptococcal glomerulonephritis
Otitis media
Poststreptococcal reactive arthritis
Mastoiditis
Paediatric autoimmune neuropsychiatric disorder
Pneumonia
associated with streptococcal infection (PANDAS)
Empyema Erysipelas
Poststreptococcal uveitis
Cellulitis
Poststreptococcal cutaneous vasculitis
Myositis/fasciitis
Septicaemia Endocarditis Toxic shock syndrome GAS, group A streptococcus.
or streptolysins including streptolysin O, which is oxygen-labile. Streptolysin O acts as an antigen triggering specific B cells to produce antistreptolysin O (ASO) antibodies. These antibodies do not have a protective function.4 Studies of the natural history of the antibody response following GAS infection have shown that the titre begins to increase within 1 week and reaches a peak at 3–6 weeks after onset of infection.9 Following this, there is a gradual decrease in titre. This is the ‘classic’ immune response. Johnson et al performed a prospective study in which throat swabs were obtained every 4 weeks and serum samples every 13 weeks from 160 subjects over a 2-year period.10 They were able to correlate changes in throat pathogens with ASO and anti-DNase B titres with history and examination findings. They identified several variations from the ‘classic’ immune response that can confound interpretation, particularly of a single measurement of ASOT. These variations include a rise in only one of the antistreptococcal antibodies, delayed decrease in antibody titre following infection and persistently elevated titres associated with a carrier state. TECHNOLOGICAL BACKGROUND (HOW IS ASOT MEASURED?) The initial method for measuring ASO antibodies was developed by Todd in 1932.5 It was based on the observation that streptolysin O lyses red blood cells and that this effect can be blocked by ASO antibodies. Todd assayed sera from patients who had recently had a GAS infection and compared this with control subjects. He noted that sera from patients were able to
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neutralise streptolysin O activity at relatively high dilutions compared with controls. The original method for measuring ASOT uses the haemolysis neutralisation assay. This is well standardised and more comparable between laboratories compared with more recent tests, including latex agglutination, nephelometry and turbidometry.11 12 The haemolysis neutralisation assay is described in figure 1.13 If a series of serum samples are available from the same patient, they should be tested simultaneously to minimise errors resulting from interassay variation.3 The change in titre between acute and convalescent measurements can be expressed as either ‘fold change’ or increase in log10 units. For example, an increase from 100 to 400 IU is a fourfold change or an increase by 0.6 log10 units (ie, log10 400–log10 100). Precautions should be taken when performing the test to avoid obtaining false ASOT measurements. As mentioned above, streptolysin O is oxygen-labile, therefore testing is under reducing conditions in the presence of sulfhydryl compounds. Inactive streptolysin O in the assay would give a falsely high titre and can be avoided by using fresh streptolysin maintained in the reduced state. Use of lipaemic, old or contaminated serum may give falsely high ASO titres because the sera could contain a lipoprotein that acts as an inhibitor of streptolysin O. This can be avoided by using fresh serum and removing the lipoprotein inhibitor with dextran sulfate.5 Falsely elevated ASOT is also found in patients whose sera contain high rheumatoid factor and those with hypergammaglobulinaemia or multiple myeloma.4 INDICATIONS AND LIMITATIONS The ASOT is a relatively inexpensive and straightforward test to perform; however, difficulties in its interpretation can lead to misdiagnosis, resulting in inappropriate treatments and anxiety for children and their families. It is important to select patients for testing carefully and interpret results in the clinical context following a detailed history and examination. Prior to testing, the clinician should aim to answer the following questions: 1. Why do I want to test for ASOT in this particular patient? 2. How is the result of the test going to change the clinical management? What is a ‘normal’ value for ASOT?
There are significant difficulties assigning a ‘normal’ value to ASOT. There is certainly not a single value or range that can be applied to all children globally. First, studies have used two different definitions of the ‘upper limit of normal’ (ULN): (1) the 80th centile and (2) the value 2 SDs greater than the mean. The use of the 80th centile rather than the latter definition results from evidence that greater than 80–90% of
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Interpretations
Figure 1 Antistreptolysin O titre (ASOT) measurement using the haemolysis neutralisation assay. (1) Reduced streptolysin O antigen is prepared in distilled water to a concentration of 2 international units (IU) per ml. (2) 1 ml of sheep red blood cells are washed three times in buffer and, after centrifugation, resuspended in buffer to give an 8% cell suspension. (3) Patient serum is mixed with buffer to give the following dilutions: 1:50, 1:200, 1:300, 1:400, 1:600 and 1:800. (4) Reduced streptolysin O is added to all dilutions of serum. (5) A negative control tube containing only buffer and a positive control tube containing buffer and streptolysin O but no serum are prepared. (6) All are mixed and left at 4°C for 2 h to allow antibody-antigen complexes to form. (7) 8% sheep red blood cell suspension is added to all tubes and incubated in a water bath at 37°C for 30 min. (8) Tubes are centrifuged at 1000 g for 2 min and observed for haemolysis. (9) Negative control (buffer only) should show no haemolysis and positive control (streptolysin O with no serum) should show complete haemolysis. (10) The ASOT is the reciprocal of the highest dilution showing no sign of haemolysis and is given in Todd units or IU (if the streptolysin O reagent is the Todd standard or international standard, respectively). In this illustration, the ASOT is 200 IU.
patients with ARF or PSGN have ASOT above the 80th centile for healthy controls without evidence of recent streptococcal infection.14 Secondly, multiple studies have shown that the ULN, however defined, is affected by the following factors:
1. 2. 3. 4. 5.
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the age of the child the site of infection geographical location season of the year use of antibiotics or steroid treatments.
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Interpretations Table 2
Average and ULN for ASOT in children reported in studies
Reference
Country
Definition of average, ULN
Season
Age group
Average
ULN
Sweden
Mean, 95% confidence limit
All
≤3 4 5 6 7–8 9 10 11 12 15–19
17 30 38 13 19 30 29 30 21 25
21 53 86 112 211 183 205 258 168 204
67 143 235 297 462 436 551 611 507 501
Kaplan16
USA
GMT, upper 20% of distribution
All
2 3 4 5 6 7 8 9 10 11 12
27 51 81 122 146 161 131 135 109 87 81
52 52 52 56 72 87 110 117 126 129 141
160 120 120 160 240 240 240 240 320 320 320
Sethi17
Chandigarh, India
GMT, GMT + 2SD
N/A
5–10 11–15
131 59
113.72 110.32
230.62 242.87
Danchin9
Melbourne, Australia
GMT, 80th centile
Autumn/winter
4–5 6–9 10–14
20 19 25
31 164 180
120 480 320
Blyth18
Sydney, Australia
GMT, 80th centile
All
0–1.9 2–3.9 4–5.9 6–7.9 8–10.9 11–14.9 15–19.9
9.2 42.5 80.7 145.6 135.1 85.2 98.8
0 0 100 200 200 200 200
Steer19
Fiji
Median, 80th centile
N/A
5 6 7 8 9 10 11 12 13 14
116 126 134 139 143 145 146 146 145 144
222 241 255 265 272 276 278 277 275 272
Mahendrappa20
Mysore, India
GMT, ULN not defined
N/A
5–10 10–15
98 102
121.8 112.5
236.3 244.5
Kotby21
Cairo, Egypt
GMT, 80th centile
Mean 9.55 years
200 N/A N/A N/A N/A
245.09 N/A N/A N/A N/A
400 400 200 200 400
Renneberg
15
All Winter Spring Summer Autumn GMT, geometric mean titre; N/A, not available; No, number of subjects; ULN, upper limit of normal.
Table 2 summarises the average and ULN from studies illustrating variation on age and location. How do I interpret a single measurement of ASOT?
A not uncommon clinical scenario is the presentation of a child with symptoms suggestive of a poststreptococcal complication, including erythematous rash, arthritis or glomerulonephritis. The child has ASOT measured and comparison made with the ULN. The 4
No
184 226 181 150 148 169 162 16 13 17 24 27 27 22 32 6 2
most obvious interpretation would be that a titre above the ULN indicates previous GAS infection and one below the ULN provides evidence against this. Blyth et al performed a retrospective study to calculate the sensitivity and specificity of ASOT interpreted in this way.18 They identified 44 subjects who developed poststreptococcal diseases between 1990 and 2005 who were seen at a tertiary hospital in Sydney, Australia. All were matched for age and sex with
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Interpretations controls. The ULN was calculated as the 80th centile of the titres of 2321 serum samples from patients who had no culture evidence of GAS in the preceding 6 months and no poststreptococcal disease (data shown in table 2). Using these subjects and controls, the authors calculated the sensitivity of ASOT using the ULN as threshold to be 72.7% (95% CI 64.2% to 77.0%) and specificity of 93.2% (95% CI 84.7% to 97.5%), respectively. Therefore, using a single measurement of ASOT alone gives a false-positive rate of 6.8% and false-negative rate of 27.3%. Blyth et al calculated that the use of a more stringent threshold for the ULN of the 95th centile (eg, equivalent to a titre of 600 for the 6-year to 10.9- year age group) gave a sensitivity and specificity of 45.5% and 100%, respectively. There are several reasons why the use of a single ASOT measurement and the ULN threshold is liable to misdiagnosis of preceding GAS infection. An elegant study by Johnson et al provides detailed evidence of the time course of changes in antistreptococcal antibodies associated with symptoms of pharyngitis and acquisition of GAS.10 They followed 160 children over a 2-year period with monthly throat cultures and serum samples taken every 13 weeks, with additional samples taken if symptomatic. The ULN for ASOT was 240, as defined in a previous study of asymptomatic children aged 2–12 years from across the USA.16 A significant rise in ASOT was defined as an increase in titre of ≥0.2 log10 between sequential specimens, and this was taken to demonstrate bona fide infection. Over the study period, 54 serum samples showed a significant increase in ASOT but 59% of these peaked below the ULN of 240. Therefore, if only a single ASOT measurement is taken and compared with the ULN, a proportion of definite GAS infections will be missed. Conversely a single measurement above the ULN can falsely diagnose GAS. In the same study, 239 of 1679 serum samples had ASOT greater than the ULN but only 23 (9.6%) corresponded to a ‘true’ infection as defined above. One reason for this is that the ASOT can remain elevated for a considerable time following GAS infection: among 24 subjects with a significant increase in ASOT during the 2-year period, 83.3% had titres above preinfection levels for > 6 months and 67.7% for > 1 year, with the majority having no evidence of reinfection.
How do I interpret two sequential measurements of ASOT?
The above findings illustrate that interpretation of a single ASOT measurement is fraught with difficulties and emphasise that the gold standard, and WHO recommendation, is that two assays are performed 10–14 days apart.13 The WHO advises that only a fourfold rise in titre between the two serological
samples should be considered as indicative of recent GAS infection.13 Even the use of sequential measurements, however, is susceptible to false positives and false negatives. Group C and group G streptococci, although not associated with the same clinical diseases, produce antigenically identical streptolysin O to GAS. They can therefore generate false positives. Johnson et al identified eight acquisitions of group C or group G streptococci, of which three (37.5%) resulted in a significant rise in ASOT.10 The mean latent periods for ARF and PSGN are 18 days and 12 days, respectively, following pharyngitis, but 2–3 weeks for PSGN following skin infection.3 In the majority of cases, the onset of disease is within 1 week to 1 month of infection. Typically, the ASOT begins to rise within 7–10 days with a peak response 2–3 weeks after the acute infection, followed by a relative plateau for 3–6 months and then a decline over the subsequent 6 months.5 22 Given these dynamics, a rise in titre is most likely to be seen if the first serum sample is taken at the time of initial presentation. Subsequent assays at weekly intervals will provide the strongest evidence of an increasing trend.3 However, it is possible that the first serum sample taken when the child presents with symptoms of a possible non-suppurative complication is already after the peak in ASOT. In this scenario, the absence of an increasing ASOT may erroneously be interpreted as excluding preceding GAS infection resulting in a false negative. A solution to these problems is the measurement of a second antistreptococcal antibody in parallel with ASOT. The most well studied and widely available is anti-DNase B. A rise in this is more specific for GAS than ASOT. Among the acquisitions of group C and group G streptococci identified by Johnson et al, none was followed by a rise in anti-DNase B.10 The time course for ADB is also different, with a rise beginning in the second week after infection and peak at 6–8 weeks.4 It is therefore more likely still to be rising at the time of presentation with symptoms of a possible poststreptococcal disease. In the diagnosis of non-suppurative complications of GAS, Blyth et al calculated that use of anti-DNase B alone, with a ULN of the 80th centile of age-matched controls, had a sensitivity and specificity of 70.5% and 93.2%, respectively.18 This was not significantly different from the use of ASOT alone. In a child with an uncomplicated acute pharyngitis, does ASOT identify those with group A streptococcal infection?
The basis of the ASOT is the generation of antibodies against streptolysin O following infection. Given the time course of ASOT discussed above, the titre at the time of acute infection would not be expected to be elevated. At the time of an acute pharyngitis, the WHO identifies the gold standard for detecting S pyogenes infection as a throat swab cultured on
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Interpretations blood agar.23 An alternative method is the rapid antigen test based on identification of a specific GAS carbohydrate cell wall antigen. Neither this nor culture is able to distinguish between true GAS infection and carrier status with a viral pharyngitis. Given the delay in rise of ASOT after the onset of GAS pharyngitis, the WHO states that “serological examination for streptococcal antibodies is not required for cases of uncomplicated streptococcal upper respiratory tract infection”.23 More recent guidelines reiterate that antistreptococcal antibody assays are not recommended for diagnosis of acute pharyngitis.24 In our opinion, if blood tests are being taken for another valid clinical indication, it may be reasonable to save a serum sample that can be assayed together with a convalescent sample if the child subsequently presents with a suspected poststreptococcal complication. Should ASOT be performed as part of a ‘rheumatic screen’?
As with any investigations, a clinician should have a clear differential diagnosis and be able to interpret the results of a test before requesting it. If the differential includes a poststreptococcal complication, it is reasonable to perform ASOT. If the prior probability is low, however, performing any form of ‘screen’ is likely to generate false positives with the consequence of further testing and increased anxiety for the family. Should ASOT be performed in every child presenting with a possible non-suppurative complication from GAS infection?
When making a diagnosis of non-suppurative complications of GAS, history and examination are insufficient to identify preceding streptococcal infection for several reasons: 1. the precipitating infection usually precedes presentation by which time the symptoms and signs may have resolved; 2. a preceding history of infection (eg, sore throat) may be affected by recall bias; 3. a sore throat may be caused by viruses or bacteria other than GAS; 4. a preceding infection may be asymptomatic.
Currently the only way to confirm previous infection is by serology. In such situations, we would recommend testing both ASOT and anti-DNase B, as discussed in more detail below. Can I use ASOT testing to make a diagnosis of acute rheumatic fever (ARF)?
The diagnostic criteria for ARF were revised in 1992 and are summarised in box 1. The WHO recommends that streptococcal serum antibody tests, of which ASOT is one, be undertaken for all suspected cases of ARF at the time of initial presentation and 3–4 weeks later.23 Together with two major, or one major and two minor, criteria, a rising ASOT can be used to make a 6
Box 1 Diagnostic criteria for acute rheumatic fever (ARF)23 Primary episode of ARF ▸ Two major criteria OR one major and two minor criteria AND ▸ Evidence of a preceding group A streptococcal infection within the last 45 days Major criteria ▸ Carditis ▸ Polyarthritis ▸ Chorea ▸ Erythema marginatum ▸ Subcutaneous nodules Minor criteria ▸ Fever, polyarthralgia ▸ Elevated acute phase reactants (erythrocyte sedimentation rate or leucocyte count) ▸ Electrocardiogram: prolonged P-R interval Evidence of preceding GAS infection: ▸ Elevated or rising antistreptolysin-O or other streptococcal antibody, OR ▸ A positive throat culture, OR ▸ Rapid antigen test for group A streptococci, OR ▸ Recent scarlet fever
diagnosis of ARF. However, difficulties often arise because only a single ASOT result is available. As has been noted above, there are problems with trying to define a meaningful ‘upper limit of normal’ for ASOT. The ASOT may be below the ULN in up to 20% of patients with a diagnosis of ARF;8 therefore, it should not be used alone to exclude a preceding GAS infection and thereby exclude ARF.25 However, by undertaking more than one streptococcal antibody test, 92– 98% of patients with ARF will have elevation of at least one.4 Among patients who have had GAS infection, approximately 80% will have a measurable rise in titre if antibodies to a single streptococcal extracellular antigen are measured. If the response to several antigens is assessed, 95% of patients show a rise in at least one.14 In a retrospective study of ASOT, anti-DNase B and antistreptokinase results from patients with poststreptococcal disease and normal age-matched controls, Blyth et al were able to demonstrate that the addition of anti-DNase B to ASOT increased sensitivity from 72.7% to 95.5%, with little loss of specificity from 93.2% to 88.6%.18 Importantly, this reduces the false-negative rate from 27.3% to 4.5%. Combination with a third antibody did not significantly improve sensitivity or specificity. By comparing different options, Blyth et al identified that the combination of ASOT and anti-DNase B, with either being positive (ULN defined as 80th centile), resulted in the highest sensitivity and specificity.
Sen ES, et al. Arch Dis Child Educ Pract Ed 2014;0:1–8. doi:10.1136/archdischild-2013-304884
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Interpretations Clinical bottom line ▸ Testing for ASOT is not recommended for diagnosis of uncomplicated acute pharyngitis ▸ A four-fold rise in ASOT between two samples taken 10-14 days apart is evidence of preceding streptococcal infection ▸ The definition of elevated ASOT should be made by comparison with age-matched controls in the same geographical location ▸ “Negative” ASOT cannot exclude ARF or PSGN ▸ Measurement of anti-DNase B, a second streptococcal serological marker, together with ASOT is recommended to improve sensitivity
In patients with acute glomerulonephritis, does a ‘normal’ ASOT exclude poststreptococcal aetiology
PSGN is a relatively frequent cause of acute glomerulonephritis in children with an estimated 404 000 new cases annually worldwide.26 The majority of cases are preceded by either streptococcal pharyngitis or skin infection.27 Among a group of 37 children with PSGN in a retrospective study from Sydney, Australia, 29 (78.4%) had an elevated ASOT using the ULN as the 80th centile of the normal population.28 Therefore, normal ASOT does not exclude PSGN. Through comparison with age-matched and sex-matched controls, the authors calculated the sensitivity and specificity of ASOT for diagnosis of PSGN as 78.4% and 89.2%, respectively. When tested for ASO, anti-DNase B and antistreptokinase, at least one was elevated in 35 (94.6%) of the patients. Analysis of each antibody if used individually did not show a statistically significant difference compared with the other two.28 However, ASOT is higher in pharyngitis-associated PSGN, whereas preceding skin infection is associated with higher anti-DNase B titres.27 The poor response, and therefore lower sensitivity, of ASOT following skin infection is thought to be due to free cholesterol in the skin binding to streptolysin O and reducing its antigenicity.29 Topics for further research
The recent study by Johnson et al examining the time course of antistreptococcal antibodies in relation to symptoms and infection highlights significant heterogeneity in response between individuals.10 In particular, although ASOT may begin to decline 6–8 weeks after initial infection, in a significant number of subjects relatively high levels persist for over 12 months. The result is that if a single ASOT is measured, the presenting symptoms may wrongly be attributed to a recent GAS infection with subsequent misdiagnosis. It is unclear why high titres are maintained even without evidence of reinfection. A better understanding of the
host response and its variation would aid in interpretation of ASOT results. An ideal marker of preceding GAS infection would be one that changes in a timely and predictable fashion following infection, that responds specifically to GAS and that distinguishes clearly between true infection and carriage. While advances have taken place in the diagnosis of acute GAS pharyngitis using quantitative polymerase chain reaction (qPCR),30 our ability to identify infection several weeks earlier remains dependent on serology. Are there other antistreptococcal antibodies, or combinations of these, that can identify preceding GAS infections with higher sensitivity and specificity and with dynamics that allow better dating of infection with respect to presenting symptoms? The literature search strategy for this article and multiple choice questions are included in the online supplementary appendix. Contributors ESS drafted and revised the paper. AVR revised the draft paper. Competing interests None. Provenance and peer review Commissioned; externally peer reviewed.
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Interpretations 12 Gerber MA, Caparas LS, Randolph MF. Evaluation of a new latex agglutination test for detection of streptolysin O antibodies. J Clin Microbiol 1990;28:413–15. 13 Vandenpitte J, Verhaegen J, Engbaek K, et al. Basic laboratory procedures in clinical bacteriology. Geneva: World Health Organisation, 2003. 14 Ayoub EM, Wannamaker LW. Evaluation of the streptococcal deoxyribonuclease B and diphosphopyridine nucleotidase antibody tests in acute rheumatic fever and acute glomerulonephritis. Pediatrics 1962;29:527–38. 15 Renneberg J, Söderström M, Prellner K, et al. Age-related variations in anti-streptococcal antibody levels. Eur J Clin Microbiol Infect Dis 1989;8:792–5. 16 Kaplan EL, Rothermel CD, Johnson DR. Antistreptolysin O and anti-deoxyribonuclease B titers: normal values for children ages 2 to 12 in the United States. Pediatrics 1998;101:86–8. 17 Sethi S, Kaushik K, Mohandas K, et al. Anti-streptolysin O titers in normal healthy children of 5–15 years. Indian Pediatr 2003;40:1068–71. 18 Blyth CC, Robertson PW. Anti-streptococcal antibodies in the diagnosis of acute and post-streptococcal disease: streptokinase versus streptolysin O and deoxyribonuclease B. Pathology 2006;38:152–6. 19 Steer AC, Vidmar S, Ritika R, et al. Normal ranges of streptococcal antibody titers are similar whether streptococci are endemic to the setting or not. Clin Vaccine Immunol 2009;16:172–5. 20 Mahendrappa KB, Rajendra . Upper limit of normal antistreptolysin-O titer in healthy school children. Indian Pediatr 2010;47:629.
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21 Kotby AA, Habeeb NM, Ezz El Elarab S. Antistreptolysin O titer in health and disease: levels and significance. Pediatr Rep 2012;4:e8. 22 Martins TB, Veasy LG, Hill HR. Antibody responses to group A streptococcal infections in acute rheumatic fever. Pediatr Infect Dis J 2006;25:832–7. 23 World Health Organisation. Rheumatic fever and rheumatic heart disease: report of a WHO expert consultation. Geneva: World Health Organisation, 2004. 24 Shulman ST, Bisno AL, Clegg HW, et al. Clinical practice guideline for the diagnosis and management of group A streptococcal pharyngitis: 2012 update by the Infectious Diseases Society of America. Clin Infect Dis 2012;55:e86–102. 25 Wannamaker LW, Ayoub EM. Antibody titers in acute rheumatic fever. Circulation 1960; 21:598–614. 26 Carapetis JR, Steer AC, Mulholland EK, et al. The global burden of group A streptococcal diseases. Lancet Infect Dis 2005;5:685–94. 27 Eison TM, Ault BH, Jones DP, et al. Post-streptococcal acute glomerulonephritis in children: clinical features and pathogenesis. Pediatr Nephrol 2011;26:165–80. 28 Blyth CC, Robertson PW, Rosenberg AR. Post-streptococcal glomerulonephritis in Sydney: a 16-year retrospective review. J Paediatr Child Health 2007;43:446–50. 29 Kaplan EL, Wannamaker LW. Suppression of the antistreptolysin O response by cholesterol and by lipid extracts of rabbit skin. J Exp Med 1976;144:754–67. 30 Dunne EM, Marshall JL, Baker CA, et al. Detection of group a streptococcal pharyngitis by quantitative PCR. BMC Infect Dis 2013;13:312.
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References
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Education & Practice Online First, published on January 30, 2014 as 10.1136/archdischild-2013-304884 INTERPRETATIONS
How to use antistreptolysin O titre E S Sen, A V Ramanan
▸ The literature search strategy for this article and multiple choice questions are included in the online supplementary appendix. To view these files please visit the journal online (http://dx.doi.org/10.1136/ archdischild-2013-304884). Department of Paediatric Rheumatology, Bristol Royal Hospital for Children, Bristol, UK Correspondence to Dr ES Sen, Department of Paediatric Rheumatology, Bristol Royal Hospital for Children, Upper Maudlin Street, Bristol, BS2 8BJ, UK; [email protected] Received 15 September 2013 Revised 14 December 2013 Accepted 3 January 2014
To cite: Sen ES, Ramanan AV. Arch Dis Child Educ Pract Ed Published Online First: [ please include Day Month Year] doi:10.1136/archdischild2013-304884
ABSTRACT Group A streptococcus (GAS) is the cause of a wide range of acute suppurative and, following a latent period, non-suppurative diseases such as rheumatic fever and poststreptococcal glomerulonephritis. Diagnosis of the latter group requires evidence of preceding GAS infection. The bacteria produce a range of extracellular antigens, including streptolysin O, which induce an antibody response in the host. A rise in antistreptolysin O titre (ASOT) is indicative of preceding GAS infection. In clinical practice, often only a single ASOT measurement is available and its timing in relation to a possible GAS infection is unknown. Interpretation of the result in this context is liable to misdiagnosis. In order to optimise diagnosis of preceding GAS infection, at least two sequential ASOT measurements, together with simultaneous assay for anti-DNase B, a second antistreptococcal antibody, is recommended.
INTRODUCTION Group A Streptococcus (GAS; Streptococcus pyogenes) is a Gram-positive, β-haemolytic bacterium that can cause both acute suppurative disease and also non-suppurative complications in humans. The diseases caused by GAS are shown in table 1. They most commonly affect infants, children and young adults. The primary site of infection is frequently the skin or throat; indeed, streptococcal pharyngotonsillitis remains the commonest form of bacterial throat infection.3 Acute GAS infection may also present as scarlet fever, characterised by an erythematous rash starting on the head and neck before spreading to the trunk and limbs with onset 1–2 days after throat infection. There are 4–5 million cases of GAS infections each year in the USA alone, with higher incidence in developing countries.4 Non-suppurative complications of GAS infections can have significant morbidity and mortality. They include acute rheumatic fever (ARF), rheumatic heart disease (RHD), poststreptococcal
glomerulonephritis (PSGN), poststreptococcal reactive arthritis (PSRA) and paediatric autoimmune neuropsychiatric disorder associated with streptococcal infection (PANDAS). The pathogenesis of these conditions is thought to be shared epitopes on antigens from GAS and the host resulting in an autoimmune response.2 Onset of these complications is often several weeks after an acute infection when symptoms and signs may have resolved. In a significant number of cases, the initial infection is subclinical.5 Throat cultures taken at the time of presentation with rheumatic fever or nephritis may be negative because of clearance of GAS by the host immune system during the latent period5 or due to previous antibiotic treatment.6 Interpretation of throat cultures at the time of presentation with a possible poststreptococcal complication is further confounded because a swab positive for GAS may represent asymptomatic carriage and not infection. A meta-analysis of 18 studies reported the prevalence of GAS carriage among asymptomatic children to range from 3% to 26%, with a pooled prevalence of 12% (95% CI 9% to 14%).7 However, diagnosis of non-suppurative complications requires evidence of recent preceding streptococcal infection. This can be provided by serological evaluations, one of which is measurement of antistreptolysin O titre (ASOT). Other serological tests include antideoxyribonuclease B (anti-DNase B), antihyaluronidase and antistreptokinase antibodies.8 This paper aims to identify which children should have ASOT measured and address questions regarding the timing and interpretation of the results.
PHYSIOLOGICAL BACKGROUND (WHAT IS ANTISTREPTOLYSIN O?) When cultured on blood agar, GAS produces a clear area of haemolysis around its colonies. This results from secretion of proteins called streptococcal haemolysins,
Sen ES, et al. Arch Dis Child Educ Pract Ed 2014;0:1–8. doi:10.1136/archdischild-2013-304884 1 Copyright Article author (or their employer) 2014. Produced by BMJ Publishing Group Ltd under licence.
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Interpretations Table 1 Suppurative and non-suppurative presentations of GAS disease1 2 Suppurative
Non-suppurative
Tonsillopharyngitis
Acute rheumatic fever
Peritonsillar abscess
Rheumatic heart disease
Cervical lymphadenitis
Poststreptococcal glomerulonephritis
Otitis media
Poststreptococcal reactive arthritis
Mastoiditis
Paediatric autoimmune neuropsychiatric disorder
Pneumonia
associated with streptococcal infection (PANDAS)
Empyema Erysipelas
Poststreptococcal uveitis
Cellulitis
Poststreptococcal cutaneous vasculitis
Myositis/fasciitis
Septicaemia Endocarditis Toxic shock syndrome GAS, group A streptococcus.
or streptolysins including streptolysin O, which is oxygen-labile. Streptolysin O acts as an antigen triggering specific B cells to produce antistreptolysin O (ASO) antibodies. These antibodies do not have a protective function.4 Studies of the natural history of the antibody response following GAS infection have shown that the titre begins to increase within 1 week and reaches a peak at 3–6 weeks after onset of infection.9 Following this, there is a gradual decrease in titre. This is the ‘classic’ immune response. Johnson et al performed a prospective study in which throat swabs were obtained every 4 weeks and serum samples every 13 weeks from 160 subjects over a 2-year period.10 They were able to correlate changes in throat pathogens with ASO and anti-DNase B titres with history and examination findings. They identified several variations from the ‘classic’ immune response that can confound interpretation, particularly of a single measurement of ASOT. These variations include a rise in only one of the antistreptococcal antibodies, delayed decrease in antibody titre following infection and persistently elevated titres associated with a carrier state. TECHNOLOGICAL BACKGROUND (HOW IS ASOT MEASURED?) The initial method for measuring ASO antibodies was developed by Todd in 1932.5 It was based on the observation that streptolysin O lyses red blood cells and that this effect can be blocked by ASO antibodies. Todd assayed sera from patients who had recently had a GAS infection and compared this with control subjects. He noted that sera from patients were able to
2
neutralise streptolysin O activity at relatively high dilutions compared with controls. The original method for measuring ASOT uses the haemolysis neutralisation assay. This is well standardised and more comparable between laboratories compared with more recent tests, including latex agglutination, nephelometry and turbidometry.11 12 The haemolysis neutralisation assay is described in figure 1.13 If a series of serum samples are available from the same patient, they should be tested simultaneously to minimise errors resulting from interassay variation.3 The change in titre between acute and convalescent measurements can be expressed as either ‘fold change’ or increase in log10 units. For example, an increase from 100 to 400 IU is a fourfold change or an increase by 0.6 log10 units (ie, log10 400–log10 100). Precautions should be taken when performing the test to avoid obtaining false ASOT measurements. As mentioned above, streptolysin O is oxygen-labile, therefore testing is under reducing conditions in the presence of sulfhydryl compounds. Inactive streptolysin O in the assay would give a falsely high titre and can be avoided by using fresh streptolysin maintained in the reduced state. Use of lipaemic, old or contaminated serum may give falsely high ASO titres because the sera could contain a lipoprotein that acts as an inhibitor of streptolysin O. This can be avoided by using fresh serum and removing the lipoprotein inhibitor with dextran sulfate.5 Falsely elevated ASOT is also found in patients whose sera contain high rheumatoid factor and those with hypergammaglobulinaemia or multiple myeloma.4 INDICATIONS AND LIMITATIONS The ASOT is a relatively inexpensive and straightforward test to perform; however, difficulties in its interpretation can lead to misdiagnosis, resulting in inappropriate treatments and anxiety for children and their families. It is important to select patients for testing carefully and interpret results in the clinical context following a detailed history and examination. Prior to testing, the clinician should aim to answer the following questions: 1. Why do I want to test for ASOT in this particular patient? 2. How is the result of the test going to change the clinical management? What is a ‘normal’ value for ASOT?
There are significant difficulties assigning a ‘normal’ value to ASOT. There is certainly not a single value or range that can be applied to all children globally. First, studies have used two different definitions of the ‘upper limit of normal’ (ULN): (1) the 80th centile and (2) the value 2 SDs greater than the mean. The use of the 80th centile rather than the latter definition results from evidence that greater than 80–90% of
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Interpretations
Figure 1 Antistreptolysin O titre (ASOT) measurement using the haemolysis neutralisation assay. (1) Reduced streptolysin O antigen is prepared in distilled water to a concentration of 2 international units (IU) per ml. (2) 1 ml of sheep red blood cells are washed three times in buffer and, after centrifugation, resuspended in buffer to give an 8% cell suspension. (3) Patient serum is mixed with buffer to give the following dilutions: 1:50, 1:200, 1:300, 1:400, 1:600 and 1:800. (4) Reduced streptolysin O is added to all dilutions of serum. (5) A negative control tube containing only buffer and a positive control tube containing buffer and streptolysin O but no serum are prepared. (6) All are mixed and left at 4°C for 2 h to allow antibody-antigen complexes to form. (7) 8% sheep red blood cell suspension is added to all tubes and incubated in a water bath at 37°C for 30 min. (8) Tubes are centrifuged at 1000 g for 2 min and observed for haemolysis. (9) Negative control (buffer only) should show no haemolysis and positive control (streptolysin O with no serum) should show complete haemolysis. (10) The ASOT is the reciprocal of the highest dilution showing no sign of haemolysis and is given in Todd units or IU (if the streptolysin O reagent is the Todd standard or international standard, respectively). In this illustration, the ASOT is 200 IU.
patients with ARF or PSGN have ASOT above the 80th centile for healthy controls without evidence of recent streptococcal infection.14 Secondly, multiple studies have shown that the ULN, however defined, is affected by the following factors:
1. 2. 3. 4. 5.
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the age of the child the site of infection geographical location season of the year use of antibiotics or steroid treatments.
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Interpretations Table 2
Average and ULN for ASOT in children reported in studies
Reference
Country
Definition of average, ULN
Season
Age group
Average
ULN
Sweden
Mean, 95% confidence limit
All
≤3 4 5 6 7–8 9 10 11 12 15–19
17 30 38 13 19 30 29 30 21 25
21 53 86 112 211 183 205 258 168 204
67 143 235 297 462 436 551 611 507 501
Kaplan16
USA
GMT, upper 20% of distribution
All
2 3 4 5 6 7 8 9 10 11 12
27 51 81 122 146 161 131 135 109 87 81
52 52 52 56 72 87 110 117 126 129 141
160 120 120 160 240 240 240 240 320 320 320
Sethi17
Chandigarh, India
GMT, GMT + 2SD
N/A
5–10 11–15
131 59
113.72 110.32
230.62 242.87
Danchin9
Melbourne, Australia
GMT, 80th centile
Autumn/winter
4–5 6–9 10–14
20 19 25
31 164 180
120 480 320
Blyth18
Sydney, Australia
GMT, 80th centile
All
0–1.9 2–3.9 4–5.9 6–7.9 8–10.9 11–14.9 15–19.9
9.2 42.5 80.7 145.6 135.1 85.2 98.8
0 0 100 200 200 200 200
Steer19
Fiji
Median, 80th centile
N/A
5 6 7 8 9 10 11 12 13 14
116 126 134 139 143 145 146 146 145 144
222 241 255 265 272 276 278 277 275 272
Mahendrappa20
Mysore, India
GMT, ULN not defined
N/A
5–10 10–15
98 102
121.8 112.5
236.3 244.5
Kotby21
Cairo, Egypt
GMT, 80th centile
Mean 9.55 years
200 N/A N/A N/A N/A
245.09 N/A N/A N/A N/A
400 400 200 200 400
Renneberg
15
All Winter Spring Summer Autumn GMT, geometric mean titre; N/A, not available; No, number of subjects; ULN, upper limit of normal.
Table 2 summarises the average and ULN from studies illustrating variation on age and location. How do I interpret a single measurement of ASOT?
A not uncommon clinical scenario is the presentation of a child with symptoms suggestive of a poststreptococcal complication, including erythematous rash, arthritis or glomerulonephritis. The child has ASOT measured and comparison made with the ULN. The 4
No
184 226 181 150 148 169 162 16 13 17 24 27 27 22 32 6 2
most obvious interpretation would be that a titre above the ULN indicates previous GAS infection and one below the ULN provides evidence against this. Blyth et al performed a retrospective study to calculate the sensitivity and specificity of ASOT interpreted in this way.18 They identified 44 subjects who developed poststreptococcal diseases between 1990 and 2005 who were seen at a tertiary hospital in Sydney, Australia. All were matched for age and sex with
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Interpretations controls. The ULN was calculated as the 80th centile of the titres of 2321 serum samples from patients who had no culture evidence of GAS in the preceding 6 months and no poststreptococcal disease (data shown in table 2). Using these subjects and controls, the authors calculated the sensitivity of ASOT using the ULN as threshold to be 72.7% (95% CI 64.2% to 77.0%) and specificity of 93.2% (95% CI 84.7% to 97.5%), respectively. Therefore, using a single measurement of ASOT alone gives a false-positive rate of 6.8% and false-negative rate of 27.3%. Blyth et al calculated that the use of a more stringent threshold for the ULN of the 95th centile (eg, equivalent to a titre of 600 for the 6-year to 10.9- year age group) gave a sensitivity and specificity of 45.5% and 100%, respectively. There are several reasons why the use of a single ASOT measurement and the ULN threshold is liable to misdiagnosis of preceding GAS infection. An elegant study by Johnson et al provides detailed evidence of the time course of changes in antistreptococcal antibodies associated with symptoms of pharyngitis and acquisition of GAS.10 They followed 160 children over a 2-year period with monthly throat cultures and serum samples taken every 13 weeks, with additional samples taken if symptomatic. The ULN for ASOT was 240, as defined in a previous study of asymptomatic children aged 2–12 years from across the USA.16 A significant rise in ASOT was defined as an increase in titre of ≥0.2 log10 between sequential specimens, and this was taken to demonstrate bona fide infection. Over the study period, 54 serum samples showed a significant increase in ASOT but 59% of these peaked below the ULN of 240. Therefore, if only a single ASOT measurement is taken and compared with the ULN, a proportion of definite GAS infections will be missed. Conversely a single measurement above the ULN can falsely diagnose GAS. In the same study, 239 of 1679 serum samples had ASOT greater than the ULN but only 23 (9.6%) corresponded to a ‘true’ infection as defined above. One reason for this is that the ASOT can remain elevated for a considerable time following GAS infection: among 24 subjects with a significant increase in ASOT during the 2-year period, 83.3% had titres above preinfection levels for > 6 months and 67.7% for > 1 year, with the majority having no evidence of reinfection.
How do I interpret two sequential measurements of ASOT?
The above findings illustrate that interpretation of a single ASOT measurement is fraught with difficulties and emphasise that the gold standard, and WHO recommendation, is that two assays are performed 10–14 days apart.13 The WHO advises that only a fourfold rise in titre between the two serological
samples should be considered as indicative of recent GAS infection.13 Even the use of sequential measurements, however, is susceptible to false positives and false negatives. Group C and group G streptococci, although not associated with the same clinical diseases, produce antigenically identical streptolysin O to GAS. They can therefore generate false positives. Johnson et al identified eight acquisitions of group C or group G streptococci, of which three (37.5%) resulted in a significant rise in ASOT.10 The mean latent periods for ARF and PSGN are 18 days and 12 days, respectively, following pharyngitis, but 2–3 weeks for PSGN following skin infection.3 In the majority of cases, the onset of disease is within 1 week to 1 month of infection. Typically, the ASOT begins to rise within 7–10 days with a peak response 2–3 weeks after the acute infection, followed by a relative plateau for 3–6 months and then a decline over the subsequent 6 months.5 22 Given these dynamics, a rise in titre is most likely to be seen if the first serum sample is taken at the time of initial presentation. Subsequent assays at weekly intervals will provide the strongest evidence of an increasing trend.3 However, it is possible that the first serum sample taken when the child presents with symptoms of a possible non-suppurative complication is already after the peak in ASOT. In this scenario, the absence of an increasing ASOT may erroneously be interpreted as excluding preceding GAS infection resulting in a false negative. A solution to these problems is the measurement of a second antistreptococcal antibody in parallel with ASOT. The most well studied and widely available is anti-DNase B. A rise in this is more specific for GAS than ASOT. Among the acquisitions of group C and group G streptococci identified by Johnson et al, none was followed by a rise in anti-DNase B.10 The time course for ADB is also different, with a rise beginning in the second week after infection and peak at 6–8 weeks.4 It is therefore more likely still to be rising at the time of presentation with symptoms of a possible poststreptococcal disease. In the diagnosis of non-suppurative complications of GAS, Blyth et al calculated that use of anti-DNase B alone, with a ULN of the 80th centile of age-matched controls, had a sensitivity and specificity of 70.5% and 93.2%, respectively.18 This was not significantly different from the use of ASOT alone. In a child with an uncomplicated acute pharyngitis, does ASOT identify those with group A streptococcal infection?
The basis of the ASOT is the generation of antibodies against streptolysin O following infection. Given the time course of ASOT discussed above, the titre at the time of acute infection would not be expected to be elevated. At the time of an acute pharyngitis, the WHO identifies the gold standard for detecting S pyogenes infection as a throat swab cultured on
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Interpretations blood agar.23 An alternative method is the rapid antigen test based on identification of a specific GAS carbohydrate cell wall antigen. Neither this nor culture is able to distinguish between true GAS infection and carrier status with a viral pharyngitis. Given the delay in rise of ASOT after the onset of GAS pharyngitis, the WHO states that “serological examination for streptococcal antibodies is not required for cases of uncomplicated streptococcal upper respiratory tract infection”.23 More recent guidelines reiterate that antistreptococcal antibody assays are not recommended for diagnosis of acute pharyngitis.24 In our opinion, if blood tests are being taken for another valid clinical indication, it may be reasonable to save a serum sample that can be assayed together with a convalescent sample if the child subsequently presents with a suspected poststreptococcal complication. Should ASOT be performed as part of a ‘rheumatic screen’?
As with any investigations, a clinician should have a clear differential diagnosis and be able to interpret the results of a test before requesting it. If the differential includes a poststreptococcal complication, it is reasonable to perform ASOT. If the prior probability is low, however, performing any form of ‘screen’ is likely to generate false positives with the consequence of further testing and increased anxiety for the family. Should ASOT be performed in every child presenting with a possible non-suppurative complication from GAS infection?
When making a diagnosis of non-suppurative complications of GAS, history and examination are insufficient to identify preceding streptococcal infection for several reasons: 1. the precipitating infection usually precedes presentation by which time the symptoms and signs may have resolved; 2. a preceding history of infection (eg, sore throat) may be affected by recall bias; 3. a sore throat may be caused by viruses or bacteria other than GAS; 4. a preceding infection may be asymptomatic.
Currently the only way to confirm previous infection is by serology. In such situations, we would recommend testing both ASOT and anti-DNase B, as discussed in more detail below. Can I use ASOT testing to make a diagnosis of acute rheumatic fever (ARF)?
The diagnostic criteria for ARF were revised in 1992 and are summarised in box 1. The WHO recommends that streptococcal serum antibody tests, of which ASOT is one, be undertaken for all suspected cases of ARF at the time of initial presentation and 3–4 weeks later.23 Together with two major, or one major and two minor, criteria, a rising ASOT can be used to make a 6
Box 1 Diagnostic criteria for acute rheumatic fever (ARF)23 Primary episode of ARF ▸ Two major criteria OR one major and two minor criteria AND ▸ Evidence of a preceding group A streptococcal infection within the last 45 days Major criteria ▸ Carditis ▸ Polyarthritis ▸ Chorea ▸ Erythema marginatum ▸ Subcutaneous nodules Minor criteria ▸ Fever, polyarthralgia ▸ Elevated acute phase reactants (erythrocyte sedimentation rate or leucocyte count) ▸ Electrocardiogram: prolonged P-R interval Evidence of preceding GAS infection: ▸ Elevated or rising antistreptolysin-O or other streptococcal antibody, OR ▸ A positive throat culture, OR ▸ Rapid antigen test for group A streptococci, OR ▸ Recent scarlet fever
diagnosis of ARF. However, difficulties often arise because only a single ASOT result is available. As has been noted above, there are problems with trying to define a meaningful ‘upper limit of normal’ for ASOT. The ASOT may be below the ULN in up to 20% of patients with a diagnosis of ARF;8 therefore, it should not be used alone to exclude a preceding GAS infection and thereby exclude ARF.25 However, by undertaking more than one streptococcal antibody test, 92– 98% of patients with ARF will have elevation of at least one.4 Among patients who have had GAS infection, approximately 80% will have a measurable rise in titre if antibodies to a single streptococcal extracellular antigen are measured. If the response to several antigens is assessed, 95% of patients show a rise in at least one.14 In a retrospective study of ASOT, anti-DNase B and antistreptokinase results from patients with poststreptococcal disease and normal age-matched controls, Blyth et al were able to demonstrate that the addition of anti-DNase B to ASOT increased sensitivity from 72.7% to 95.5%, with little loss of specificity from 93.2% to 88.6%.18 Importantly, this reduces the false-negative rate from 27.3% to 4.5%. Combination with a third antibody did not significantly improve sensitivity or specificity. By comparing different options, Blyth et al identified that the combination of ASOT and anti-DNase B, with either being positive (ULN defined as 80th centile), resulted in the highest sensitivity and specificity.
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Interpretations Clinical bottom line ▸ Testing for ASOT is not recommended for diagnosis of uncomplicated acute pharyngitis ▸ A four-fold rise in ASOT between two samples taken 10-14 days apart is evidence of preceding streptococcal infection ▸ The definition of elevated ASOT should be made by comparison with age-matched controls in the same geographical location ▸ “Negative” ASOT cannot exclude ARF or PSGN ▸ Measurement of anti-DNase B, a second streptococcal serological marker, together with ASOT is recommended to improve sensitivity
In patients with acute glomerulonephritis, does a ‘normal’ ASOT exclude poststreptococcal aetiology
PSGN is a relatively frequent cause of acute glomerulonephritis in children with an estimated 404 000 new cases annually worldwide.26 The majority of cases are preceded by either streptococcal pharyngitis or skin infection.27 Among a group of 37 children with PSGN in a retrospective study from Sydney, Australia, 29 (78.4%) had an elevated ASOT using the ULN as the 80th centile of the normal population.28 Therefore, normal ASOT does not exclude PSGN. Through comparison with age-matched and sex-matched controls, the authors calculated the sensitivity and specificity of ASOT for diagnosis of PSGN as 78.4% and 89.2%, respectively. When tested for ASO, anti-DNase B and antistreptokinase, at least one was elevated in 35 (94.6%) of the patients. Analysis of each antibody if used individually did not show a statistically significant difference compared with the other two.28 However, ASOT is higher in pharyngitis-associated PSGN, whereas preceding skin infection is associated with higher anti-DNase B titres.27 The poor response, and therefore lower sensitivity, of ASOT following skin infection is thought to be due to free cholesterol in the skin binding to streptolysin O and reducing its antigenicity.29 Topics for further research
The recent study by Johnson et al examining the time course of antistreptococcal antibodies in relation to symptoms and infection highlights significant heterogeneity in response between individuals.10 In particular, although ASOT may begin to decline 6–8 weeks after initial infection, in a significant number of subjects relatively high levels persist for over 12 months. The result is that if a single ASOT is measured, the presenting symptoms may wrongly be attributed to a recent GAS infection with subsequent misdiagnosis. It is unclear why high titres are maintained even without evidence of reinfection. A better understanding of the
host response and its variation would aid in interpretation of ASOT results. An ideal marker of preceding GAS infection would be one that changes in a timely and predictable fashion following infection, that responds specifically to GAS and that distinguishes clearly between true infection and carriage. While advances have taken place in the diagnosis of acute GAS pharyngitis using quantitative polymerase chain reaction (qPCR),30 our ability to identify infection several weeks earlier remains dependent on serology. Are there other antistreptococcal antibodies, or combinations of these, that can identify preceding GAS infections with higher sensitivity and specificity and with dynamics that allow better dating of infection with respect to presenting symptoms? The literature search strategy for this article and multiple choice questions are included in the online supplementary appendix. Contributors ESS drafted and revised the paper. AVR revised the draft paper. Competing interests None. Provenance and peer review Commissioned; externally peer reviewed.
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How to use antistreptolysin O titre E S Sen and A V Ramanan Arch Dis Child Educ Pract Ed published online January 30, 2014
doi: 10.1136/archdischild-2013-304884
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References
This article cites 27 articles, 9 of which can be accessed free at: http://ep.bmj.com/content/early/2014/01/30/archdischild-2013-304884.full.html#ref-list-1
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