Different Serological Techniques For Detection Of Biology Essay

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Introduction: Streptococcus pyogenes is of great medical importance as it causes various diseases in children as well as adults.

What actually calls for concern in our country is the post streptococcal immunological disorders in the form of rheumatic fever and glomerulonephritis which may develop 3-4 weeks later. Around 7% of strept. infections develop rheumatic fever and the problem of renal failure is on increase.

Aim of the work: In recent years, there have been important contribution to the understanding of streptococcal infection, diagnosis, rapid evaluation for fear of its sequelae.

Methods: Streptococal infections for carriers & patients are looked for rapid serological methods which include:

- Antistreptolysin O (tube method) (ASO) and by strip method (ASL).

- Antistreptodoronas method for titration of anti Dnase B.

- Antibodies to extracellular antigens of streptococcus i.e. more than one antibody which is called "streptozyme" is done.

Results: * Evaluation of the 4 serological methods was done in this study with sensitivity and specificity.

* It was found that the "Streptozyme" methods is superior, rapid, valuable since it is less time consuming (2minutes), utilizes serum, plasma activated or non and fingertip blood.

* Results are not affected by hemolysis, elevated cholesterol or anticoagulants which can affect ASO tests.

Conclusion: It is suitable test for screening and titration.


Pathogenic streptococci that cause infections are beta haemolytic. The structure of the bacterial cell (i.e. the hyaluronic acid capsule, specific surface antigens, peptidoglycan cell wall) is important factors in pathogenesis.

Many of the extracellular products are also important, including exotoxins, DNAS, haemolysins (strepto-lysin O and S) hyaluronidase and strepto kinase which are so valuable now in rapid diagnosis of streptococcus infections.(1,2)

Exotoxins are responsible for the rash of scarlet fever and also produce fever and damage the reticulo endothelial response.(1)

Group A streptococci commonly cause bacterial pharyngitis and tonsillitis(1) and may also be implicated in lower respiratory infections in communities and travellers,(3) puerperal sepsis, infections of burns, skin sepsis specially in hot climatic areas,(4) cellulitis, impetigo, erysipelas, endocarditis,(5) pericarditis, meningitis, Necrotising fasciitis, toxic shock syndrome(6) and epiglossitis(7)

Non purulent sequelae of streptococcal infection:

a) Post strepto-coccal glomerulonephritis which

is immunologically mediated with hypo-complementaemia.(1,8). IgA Nephropathy may be triggered by streptococcal infection,(9) Tonsillar hyper production of poly IgA in recurrent tonsillitis has been associated with IgA nephropathy.(10)

b) Rheumatic fever which is worldwide and the rheumatic damage is immunologically mediated, characterized by exudative lesions of connective tissues specially the heart, joints, blood vessels and subcutaneous tissues.(11)

The disease initially affect children 6-15 years

of age occurring about 3 weeks after pharyngitis.(1) Poly-arthritis occurs in 75% of patients, myocarditis in 40-50% sydenhans chorea in 15% subcutaneous and erythema marginatum are uncommon. Carditis, an important risk factor in heart diseases(12) was present in 40% of rheumatic fever attack in northern India 1993(13) and in 34% Saudi Arabia(14) in King Abdulaziz University hospital (Jeddah) 1992.

Infection with streptococcus pyogenes (B-Hemolytic) with its sequelae of arthritis; account for about 15% of arthritis (15) and carditis and the long term complication which is the valvular heart disease,(1,4) Rheumatic fever (RF) and rheumatic heart disease RHD in Egypt was 10/1000 in age group 6-12 years.(4)

Death form RHD was 13.2/100.000 in 1992 according to WHO statistic in Egypt. RHD and surgery in University hospitals and different institutions in Egypt reach 50-60% as complications of RF in 1993.

- Developing countries have extremely low health budgets, many spending less than 53 per head

on health services annually.(16) So good management and rapid diagnosis of Rheumatic diseases by well motivated community health workers is essential.(11,15) Rapid antigen detection kits for use at the bedside have become available. Kits for strep. Group A (Beta-hemolytic in throat swabs and serum are generally specific > 95%, (17-19) but sensitivity varies form 60-90% and is lower than that of throat swab culture.(17,19)

- The rapid tests are a useful help in diagnosis, treatment and play a role in limiting the spread of infection specially in catastrophic illness as in Necrotizing fasciitis.(20) And in the survey to detect the carrier of streptococcus. The carriership of haemolytic streptococci group A in Egypt by El Kholy(19) was 15% and in Italian population was 26.6% in a study of 865 children 1992.(21)

Diagnosis of Rheumatic fever: 1st, by clinical examination with the laboratory tests:

1- Leucocytic count.

2- Erythorcyte sedimentation rate, (E.S.R).

3- C.R.P. C-reactive protein which is raised as acute phase protein in RF due to strep. Pyogenes and it plays an important role in carditis, so considered risky in heart disease where it increases with interleukin 6 (cytokine secreted by the liver).(22)

So understanding the mechanism of its increase is important in treatment and prevention of heart diseases.

4- Throat swab for culture, although it is a standard method yet some important factors should be considered:

a) Bacterial flora on surface of tonsils and deep seated one.

b) Evolution of B-lactamase producing micro-organisms.(22, 24)

So difficulty in distinguishing the causative agent or agents. As result, the use of pharyngeal culture with the aim of identifying the pathogenic agents appears to be unreliable method.(23.24)

5- The Rapid serological Methods for detection of antibodies (abs) in serum or blood against synthetic exotoxins or extracellular products of streptococcus B-haemolytic.

In this study 4 serological methods available as kits form Biomerieux and Wampole Laboratories Diagnosis evaluation of the patients and methods were done.


Clinically diagnosed fifteen children aged 6-15 years with rheumatic fever with or without tonsillitis, attending the pediatric and E.N.T. departments in Alex. University.

Throat swabs were taken, inoculated on blood agar plates which were incubated at 37°C in presence of 5-10% CO2.

B-haemolytic colonies were tested for bacitracin sensitivity. The inhibited colonies diagnosed

and confirmed by sero typing as group "A" streptococci.

Serum was taken from the 15 children with rheumatic fever and 4 methods for detection of different antibodies to streptococcus group A were performed - the 4kits were:

1- Antistreptolysine "O" titration (tube method): ASO:

Principle: B-haemolytic group A streptococci produce the streptolysin "O" enzyme which in its reduced state acts as haemolysin. It stimulates the formation of ASO antibodies which can be revealed by neutralizing the enzyme activity in regard to rabbit red cells.


1-0.1 ml serum was taken, dil. 1:50 after inactivation at 56°C for 30 minutes serial dilutions in 7 tubes was done.

2- A fixed volume of ASLO was added, incubated for 15' min. at 37°C.

3- Equal volume of 5% rabbit RBCs was added to each tube.

4- Incubated for 45 minutes at 37°C.

5- Centrifuged for 2 minutes at 500g

6- Haemolysis was looked for.

Red cell control: No haemolysis

Serum titre: reverse of highest dil. giving haemolysis N<200 U/ml and more >200 denote recent infection.

2- ASL kit (Biomerieux): Titration of ASLO by rapid test in "strips"

Principle: Neutralisation by Ab in the serum sample of the haemolytic activity of streptolyoin O produced by B. haemolytic streptococcus present in the dehydrated form, in increasing amounts in the bottom of the strip wells.

Haemolytic activity is demonstrated by addition of rabbit red cells. Reagents has been standardized against a WHO reference.

Sample: Fresh or stored sera at - 20°C inactivated or not.

Turbid, lipemic, and haemolysed sera were excluded.

1- With the reducing agent available the serum was dil. 7 dilutions in the kit (10 µl serum + 1 ml DTT reagent).

2- 75 µl of dil. serum was dispensed into each well of the strip - shaken - incubated 15 minutes at room temp.

3- 75 µl of dil. rabbit RBC's (2%) was dispensed to each well.

4- Incubated 45', then centrifuged 2' at 2000 rpn.

Reading: Control: No hemolysis

Serum titre: last well with no haemolysis.

Interpretation: 200 IU/ml is considered as upper limit of normal.

3- ASD kit (Biomerieux):

Principle: Neutralization: by ab in the serum sample of the depolymerizing activity of DNase produced by group "A" streptococci, present in dehydrated form, in increasing amounts in the bottom of the strip wells.

The depolymerisation of the substrate DNA is demonstrated by a colour indicator which form blue to pink colour.

Interpretation: Anti DNas B Titre> 200u/ml in adult and >300u/ml:school age children are significant.

Anti DNase B titre generally increase later than that of ASLO, it is maintained for longer time. So anti DNase B and ALSO are complementary and if determined simultameously increase the reliability of diagnosis by 90%.

4- Streptozyme: (Wampole Laboratories):

Principle: The streptozyme reagent consists of a standardized susupension of aldhyde-fixed sheep cells sensitized with group A streptococcus extacellular antigens including some of the classical exoenzymes, such as streptolysin, streptokinase, hyaluronidase, DNase and NADase which will react with antibodies to these antigens to give a and NADase which will react with antibodies to these antigens to give a positive agglutination reaction.


- Fresh or inactivated serum or plasma, as well as peripheral blood from fingertip or earlobe was used.

- Sample 1: 100 (0.1 ml serum +9.9ml saline) was diluted.

- The capillary to mark 50ul was filled with the dil. sample.

- A section of slide was expelled.

- Drop of the reagent, was added and mixed with stirrer.

- Rocked for 2 minutes at 8-10 times per minute, observed for agglutination within 10 seconds.

- Different dilutions was prepared and repeated as in the test, the last dil. on the slide to show positive agglutination was taken as STZ titre.

- Interpretation: N: up to 100 STZ unit.

Statistical Methods:-

Validating parameters were calculated namely sensitivity and specificity.

When sensitivity = the ability of the test to detect those with the condition.

Specificity = the ability of the test to exclude those without the condition.


The 4 serological kits for estimation of antibodies to exotoxins or extra cellular enzymes were done with different dilutions:

1- ASLO (Tube Method):

The test was positive in all the 15 patient suffering form RF.

Comparing to the standard throat culture and confirmation with the clinical signs, it showed 100% sensitivity and 100% specificity.

60% of patients showed 4 fold increase.

33.3% of patients showed 8 fold increase.

6.7% of patients showed 3 fold increase.

2- ASL antistrepto lysine O titre by (strip method):

The test showed 100% sensitivity.

6.7% of patients showed 2 fold increase.

26.65% of patients showed 3 fold increase.

26.65 showed 4 fold increase.

40% showed 6 fold increase.

The above 2 methods agreed and confirmed each other.

The ASL O by the strip showed lower titres than that of the tube method. The dehydrated form may contribute to the lower titre while the tube with the chance of dilution by the ASLO buffer may allow more freedom to the mobility of the Ab.

3- ASD (strip): antistrept Doromas or anti DNas B ab evaluation showed sensitivity 93.3% and specificity 100%.

13.3 showed one fold increase.

20% showed 2 fold increase.

13.3 % showed 2.6 fold increase.

46.7% showed 5.3 fold increase.

6.7% negative for the anti DNase B extracellular enzyme of streptococcus B hemolytic.

The titre as percentage compared with ASLO titre is lower generally that may be due to the delay of appearance of the DNase before the ASLO.

4- STZ: antiextracellular enzymes (5 enzymes) of the streptolysin O and DNase strepto kinase, holuronidase and NADase.

All cases showed positive titre.

6.7% showed one fold increase.

6.7% showed two fold increase.

60% showed 4 fold increase

6.6% showed 6 fold increase.

20% showed more the 8 fold increase

The 4 methods agree in 20% high positive (case 7,9,10), the older children.

The negative patient by anti DNae, also showed only 100STZ unit i.e. upper limit of normal, while high titre with ASLO (6 and 8 fold). That agree with the statement of early appearance of ASLO exoenzyme.

Comparison of the different methods is shown in table II.


(1) The biological fluid with its recommendation.

(2) The incubation time.

(3) The stability of the reagents.

(4) The sensitivity and specificity.

ï‚· STZ method is rapid, less time consuming, accurate, and stable.

ï‚· Lipenic or turbid, hemolysed sample do not affect the sensitivity or specificity of the test.

Table I: The result of different serological techniques for detection of antibodies to streptococci

Case No.





stik method strip

ASD (strip) anti. Strept doronas

(anti DNA's B)

STZ strep to zyme anti extra cellular Enz.

n. up to 200


n. up to 200 IU/UL

N. Adult to 200 IU/UL

Children 300 IU/UL

N. up to 100 stz unit












































































Table II: Properties of the four serological methods for detection of antibodies to streptococci

Method and manufacturer

Biological fluid in the test







ASLO (Tube) Biomerieux

100 U serum inactive

30` at 56c

90 mint

1 week

<200 Iu



ASL (strip)


10 UL inactiverd or not

60 mint

1 month

at 2-8c

<200 Iu



ASD (strip)


10 UL inactiverd

150 mint

12 days

<200 u adult <300u school children




(wampole laboratories)

100 U

- Serum

- Plasma

- Finger tip blood

2 mint

Up to exp. date


STZ units




The 4 serological methods are capable to recognize a specific immune response against group A-hemolytic streptococcus strains and are valuable tools in diagnosis of rheumatic fever.

The ASLO titre and STZ (to strept products SP) were high in the older with previous history of RF attacks. The lower titre with the younger children below 12 years might be carriers or with post streptococcal attacks of tonsillitis. That agreed with Mexico study 1995.(25)

The 4 fold increase in ASLO and anti DNase titre in this study is comparable with the "OSLO" study.(26)

ASLO increase in addition to RF, in other conditions as endocarditis (5) in zones with greater air pollution and in summer evidencing toxic elements of photo oxidants,(27) and that comparable with the take in summer and autumn in our study.

It also increases in guttate psoriasis(28) and in Henoch-schonlein purpura.(29)

It was found in normal manufactured immune globulin prepared for IV therapy(30) and IgM multiple myeloma.(31)

ASLO increase in RF as a cause of scleritis, uveitis and glaucoma.(32) and in post streptococcal poly myalgia.(33)

The rise of ASLO cause reduction in the gamma globulin immunological activity.(34)

In study in Tanzania (1995), ASLO an Anti DNase B was raised 32.8% and 45.9% respectively in patients with post streptococcal pyogenes infections.(35)

In Cairo 1989, ASLO increased more than 400, showed evidence of recent Strep, pyogenes infection.(36)

In Finland ASLO was more the 500 in patients with acute arthritis or muscle symptoms on 76 patients 1993.

It was raised in 30% of patients receiving IV(37) Strepto Kinase as thrombolytic therapy together with Strept. Kinase which remained for 4 years after.(38)

Conclusion & Recommendations

Evaluating 4 (four) serological kits of estimation of antibody titre in patients with (recent, post, carrier) streptococcus pyogenes (β hemolytic group A) against exotoxin and extracellular enzymes:

ASLO (Tube and strip) methods.

Anti DNas B (antistrept Doronas).

STZ (Streptozme) kit: Estimation of five antibodies (ab) against SLO, DNase, NADas-antihyaluronidase and streptokinase.

The accuracy of streptozyme method is usually above 95% and in this study is 100% in addition to other advantages over the other kits:

1- It is a slide test "2 minute" for maximum efficiency.

2- Utilizes: serum, plasma (fresh er inactivated) finger tip blood, in volume 50-100 µl.

3- Results are not affected by:

1) Hemolysis.

2) Elevated cholesterol.

3) Anticoagulants which can affect other tests.

4) Stable for 18 month under refrigeration (i.e., the expiry date) on opposite of others.

5) Positive and negative control available with kits.

6) Its cost is suitable.

7) Suitable for screening and titration.

8) So it is recommended for mass survey especially in school children